Alkaline phosphate-based cancer treatments

Abstract

The present invention relates to methods and compositions for preventing, treating, or reducing chemotherapy treatment-mediated side effects, including gastrointestinal (GI) side effects, comprising administering to a subject an intestinal alkaline phosphatase (IAP).

Description

[Technical field]

[0001] The present invention relates inter alia to methods of preventing and / or reducing chemotherapy-mediated gastrointestinal side effects by administration of therapeutic intestinal alkaline phosphatase.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 843,850, filed May 6, 2019, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated by reference in their entirety into this specification: Computer Readable Format Copy of Sequence Listing (Filename: "SYN-046PC_ST25.txt"; Creation Date: May 4, 2020; File Size: 84,284 bytes). [Background technology]

[0004] Chemotherapy is a common cancer treatment method that has been widely used, and various chemotherapeutic agents have been developed for cancer treatment. However, many chemotherapeutic agents have side effects, including moderate to severe side effects, which may include gastrointestinal (GI) dysfunction, such as diarrhea and / or colitis. Some chemotherapeutic agents are associated with dysbiosis of the intestinal microbiome, which is one of the main causes of intestinal mucositis. Side effects limit the application of chemotherapeutic agents.

[0005] In view of the need for improved cancer therapies and reduction in adverse side effects to enable improved cancer patient care, there is an increasing need for therapeutic compositions that prevent and / or reduce the side effects associated with the administration of various anti-cancer agents.

[0006] Alkaline phosphatase ("AP", EC 3.1.3.1) is a hydrolase enzyme that can remove phosphate groups from a variety of targets, including nucleotides and proteins. Alkaline phosphatases are found in prokaryotes as well as eukaryotes, ranging from E. coli to mammals. In particular, mammalian APs have been shown to play important roles in intestinal homeostasis, mucosal barrier function, promotion of commensal bacteria, and defense against pathogens. Mammalian APs exert properties that primarily target LPS (TLR4 agonist), flagellin (TLR5 agonist), and CpG DNA (TLR9 ​​agonist). APs also degrade intestinal luminal NTPs (e.g., ATP, GTP, etc.), which promotes the growth of favorable bacteria and reverses dysbiosis. APs may therefore find clinical use in preventing and / or reducing side effects associated with, for example, the administration of anticancer drugs. Summary of the Invention

[0007] Thus, the present invention provides a method of using intestinal alkaline phosphatase (IAP) in conjunction with chemotherapy treatment for the treatment and / or prevention of side effects of chemotherapy treatment.

[0008] In some aspects, the present invention provides a method of increasing the therapeutic effectiveness of a chemotherapeutic treatment comprising a nucleoside analog in a subject in need thereof, comprising administering to the subject an effective amount of an IAP.

[0009] In some aspects of the invention, there is provided a method of treating or preventing at least one side effect of a chemotherapeutic treatment with a nucleoside analog in a subject, the method comprising administering to the subject an intestinal alkaline phosphatase (IAP), wherein the subject has undergone or is undergoing cancer treatment using a chemotherapeutic treatment.

[0010] The IAP may be administered sequentially, simultaneously, or intermittently with the administration of the nucleoside analog. The IAP may be administered orally. In some embodiments, the nucleoside analog may be a purine analog or a pyrimidine analog, or a derivative thereof. Non-limiting examples of nucleoside analogs include 5-fluorouracil, 5'-deoxyfluorouridine, 5-fluoro-2'-deoxyuridine triphosphate, fluorouridine, 5-fluoro-2'-deoxycytidine, luoropyrimidine, 2'-deoxyfluorouridine, fluorocytosine, trifluoro-methyl-2'-deoxyuridine, cyclocytidine, arabinosyl cytosine, 5-aza-2'-deoxycytidine, arabinosyl-5-azacytosine, azacytidine, 6-azacytidine, N-phosphonoacetyl-L-aspartic acid, pyrazofurin, 6-azaurate, 5 ... These include lysine, azaribine, thymidine, fazarabine, 3-deazauridine, cytarabine, gemcitabine, troxacitabine, decitabine, pseudoisocytidine, zebularine, ancitabine, capecitabine, N4-octadecyl-cytarabine, elaidic acid-cytarabine, cladribine, acyclovir, clofarabine, nelarabine, forodesine, 8-chloroadenosine, sapacitabine, thiarabine, and derivatives thereof. In an embodiment, the nucleoside analog is 5-fluorouracil.

[0011] Administration of an IAP, according to embodiments of the present disclosure, does not prevent treatment of cancer in a subject. In some aspects, administration of an IAP allows for a reduction in the duration, dose, and / or frequency of administration of a nucleoside analog. For example, in some embodiments, use of an IAP allows for the use of sub-therapeutic doses of a nucleoside analog. In some aspects, methods according to the present disclosure increase the therapeutic window of a nucleoside analog.

[0012] In some embodiments, administration of an IAP with chemotherapy treatment prevents, eliminates, or reduces side effects associated with chemotherapy treatment. Thus, in some aspects, the present disclosure provides a method for treating or preventing at least one side effect of chemotherapy treatment in a subject. The method includes administering an IAP to a subject, the subject having undergone or undergoing cancer treatment using chemotherapy treatment. Side effects of chemotherapy treatment may include one or more of diarrhea, colitis, mucositis, weight loss, pain, nausea, vomiting, constipation, anemia, malnutrition, alopecia, bone marrow suppression, nephrotoxicity, hair loss, numbness, altered taste, loss of appetite, thinned or brittle hair, mouth sores, memory loss, bleeding, cardiac toxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment. Side effects of chemotherapy treatment may also include gastrointestinal (GI) side effects, which may be caused by some chemotherapy treatments, such as nucleoside analogs (e.g., 5-fluorouracil). Methods according to the present disclosure allow for the treatment or prevention of GI side effects, including mucositis and diarrhea. [Brief description of the drawings]

[0013] [Figure 1A] Illustrates that administration of IAP reduces the side effects of 5-FU. Shown is an assessment of body weight change as the mean % body weight change ± SEM for untreated (upper curve), vehicle + 5-FU (middle curve), and SYN BIAPPII + 5-FU (lower curve) mice over days. [Figure 1B] Illustrates that administration of IAP reduces the side effects of 5-FU. Stool consistency assessment by mean stool consistency score ± SEM versus day for vehicle + 5-FU and SYN BIAPPII + 5-FU mice compared to untreated mice data shown on the x-axis baseline. As shown, administration of 5-FU caused loose stools / diarrhea and was associated with weight loss in all treated animals, which peaked on day 6 and resolved on day 9. Administration of IAP shows significantly faster recovery from loose stool / diarrhea symptoms (p<0.03). [Figure 2A]It further illustrates that administration of IAP reduces the side effects of 5-FU. Depicts mean daily weight of 50% group survival shown as percent change over time compared to day 0 weight + SEM. [Figure 2B] It is further illustrated that administration of IAP reduces the side effects of 5-FU.The results of daily stool consistency scores from days 0 to 12 are shown, with the mean scores + SEM shown. [Figure 2C] It further illustrates that administration of IAP reduces the side effects of 5-FU.Figure 1 depicts a graphical representation of the percentage of days animals had diarrhea by treatment group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention is based on the discovery that, inter alia, alkaline phosphatase (AP)-based drugs, including but not limited to intestinal alkaline phosphatase (IAP), can be used substantially together with anti-cancer treatments, including various chemotherapeutic drug treatments, such as nucleoside analogues. Based on the discovery that IAPs reduce the side effects of cancer treatments, such as chemotherapy, the inventors have discovered that the use of IAPs, which can be administered sequentially, simultaneously, or intermittently with one or more chemotherapeutic drug treatments, or in combination with one of these regimens, does not interfere with the action of the chemotherapeutic agent, much less enhance the subject's overall response to chemotherapy.

[0015] Exposure to chemotherapy drugs can result in gastrointestinal (GI) tract damage, including, but not limited to, dysbiosis, diarrhea, mucositis, leaky gut, endotoxemia, altered intestinal transit, malabsorption, and dysmotility. AP-based agents of the present invention, including, but not limited to, IAPs, optionally administered orally, can reduce or prevent these and other side effects. Thus, the present invention, in various embodiments, relates to treating or preventing the effects of chemotherapy with AP-based agents of the present invention, including, but not limited to, IAPs, which can be administered orally. The inventors have discovered that IAPs find use in preventing, reducing, or eliminating chemotherapy-mediated GI side effects, such as diarrhea and / or mucositis colitis, in patients. The use of IAPs as described above allows for more effective cancer treatment, since chemotherapy is not limited by dose- and regimen-limiting GI and other side effects.

[0016] Chemotherapeutic drug treatment involves the use of various drugs, including but not limited to nucleoside analogues, a class of antimetabolites that are widely used to treat various malignancies, including hematological cancers and solid tumors. Nucleoside analogues mimic physiological nucleosides and are incorporated into newly synthesized DNA, resulting in synthesis inhibition and chain termination. Some nucleoside analogues inhibit key enzymes involved in the production of purine and pyrimidine nucleotides, as well as RNA synthesis, and directly activate the caspase cascade. The effect of nucleoside analogue action can result in cell death. Non-limiting examples of nucleoside analogs include 5-fluorouracil, 5'-deoxyfluorouridine, 5-fluoro-2'-deoxyuridine triphosphate, fluorouridine, 5-fluoro-2'-deoxycytidine, fluoropyrimidine, 2'-deoxyfluorouridine, fluorocytosine, trifluoro-methyl-2'-deoxyuridine, cyclocytidine, arabinosyl cytosine, 5-aza-2'-deoxycytidine, arabinosyl-5-azacytosine, azacytidine, 6-azacytidine, These include N-phosphonoacetyl-L-aspartic acid, pyrazofurin, 6-azauridine, azaribine, thymidine, fazarabine, 3-deazauridine, cytarabine, gemcitabine, troxacitabine, decitabine, pseudoisocytidine, zebularine, ancitabine, capecitabine, N4-octadecyl-cytarabine, elaidic acid-cytarabine, cladribine, acyclovir, clofarabine, nelarabine, forodesine, 8-chloroadenosine, sapacitabine, thiarabine and derivatives thereof.

[0017] Most nucleoside analogs can cause potential side effects, including, but not limited to, diarrhea and / or mucositis. Other examples of side effects of chemotherapy treatment with one or more nucleoside analogs include diarrhea, colitis, mucositis, weight loss, pain, nausea, vomiting, constipation, anemia, malnutrition, alopecia, bone marrow suppression, nephrotoxicity, hair loss, numbness, altered taste, loss of appetite, thinning or brittle hair, stomatitis, memory loss, bleeding, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment.

[0018] IAPs are endogenous proteins expressed by the intestinal epithelium that promote GI homeostasis by detoxifying inflammatory mediators and may be used to maintain gut homeostasis by reducing inflammation, promoting intestinal barrier tightness, and promoting a healthy microbiome. For example, loss of IAP expression or function is associated with increased intestinal inflammation, dysbiosis, bacterial translocation, and systemic inflammation. Among other things, their main functions for maintaining gut homeostasis are generally recognized as the control of bicarbonate secretion and duodenal surface pH, absorption of long-chain fatty acids, reducing intestinal inflammation via detoxifying pathogen-associated molecular patterns, and controlling the gut microbiome. Some substrates acted upon by the phosphatase function of IAPs include, without being bound by theory, lipopolysaccharide (LPS), flagellin, CpG DNA, and nucleotide diphosphates and triphosphates. Specifically, IAPs are targets for therapeutic agents due to their ability to downregulate inflammation, control the microbiome, tighten the intestinal barrier via enhanced expression of claudins and occludins, and affect adenosine triphosphate and diphosphate (ATP and ADP) metabolism. The present invention contemplates compositions comprising IAPs that do not interfere with a patient's cancer treatment. In fact, the methods described herein according to the present invention enhance a subject's overall response to chemotherapy treatment with nucleoside analogs.

[0019] Accordingly, the present invention is directed, in part, to a method of treating or preventing at least one side effect of chemotherapeutic treatment with a nucleoside analog in a subject in need thereof, comprising administering an IAP to the subject, wherein the subject has undergone or is undergoing cancer treatment using chemotherapeutic treatment.

[0020] Alkaline phosphatase (AP) The present disclosure is directed, in part, to pharmaceutical compositions, formulations, and the use of one or more alkaline phosphatase-based drugs (AP-based drugs). Alkaline phosphatases are dimeric metalloenzymes that catalyze the hydrolysis of phosphate esters and dephosphorylate various target substrates at physiological pH and higher. Exemplary APs that can be utilized in the present invention include, but are not limited to, IAPs (e.g., calf IAP or bovine IAP, chicken IAP, goat IAP), placental alkaline phosphatase (PLAP), placental-like alkaline phosphatase, germ cell alkaline phosphatase (GCAP), tissue non-specific alkaline phosphatase (TNAP; found primarily in liver, kidney, and bone), bone alkaline phosphatase, liver alkaline phosphatase, kidney alkaline phosphatase, bacterial alkaline phosphatase, fungal alkaline phosphatase, shrimp alkaline phosphatase, modified IAP, recombinant IAP, or any polypeptide that includes alkaline phosphatase activity.

[0021] In various embodiments, the present invention contemplates the use of mammalian alkaline phosphatases, including, but not limited to, IAP, placental alkaline phosphatase (PLAP), germ cell alkaline phosphatase (GCAP), and tissue non-specific alkaline phosphatase (TNAP).

[0022] In some embodiments, the AP-based drug is an IAP. IAPs are produced in the proximal small intestine and are bound to enterocytes via a GPI anchor. Some IAPs are released into the intestinal lumen as soluble proteins that are shed from cells via phospholipases, together with vesicles shed by cells. The enzymes then traverse the small and large intestines, so that some active enzymes can be detected in stool. In some embodiments, the IAP is a human IAP (hIAP). In some embodiments, the IAP is a calf (cIAP), also known as a bovine IAP (bIAP). There are multiple isozymes of bIAP, for example, bIAP II and IV have higher specific activity than bIAP I. In some embodiments, the IAP is a cIAP or any one of the bIAP isozymes (e.g., bIAP I, II, and IV). In some embodiments, the IAP is bIAP II. In another embodiment, the IAP is bIAP IV.

[0023] Also included within the definition of IAP are IAP variants. IAP variants have at least one or more amino acid modifications, generally amino acid substitutions, compared to the parent wild-type sequence. In some embodiments, the IAPs of the present invention are at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100%, or about 101%, or about 102%, or about 103%, or about 104%, or about 105%, or about 106%, or about 107%, or about 108%, or about 109%, or about 110, or about 111, or about 112, or about 113, or about 114, or about 115, or about 116, or about 117, or about 118, or about 119, or about 120, or about 121, or about 122, or about 123, or about 124, or about 125, or about 126, or about 127, or about 128, or about 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 its biochemical activity as measured as described herein.

[0024] In various embodiments, the AP-based agent is hIAP or a variant thereof. In some embodiments, the AP-based agent is hIAP comprising the amino acid sequence of SEQ ID NO: 1, as set forth below. hIAP-SEQ ID NO:1 1 mqgpwvllll glrlqlslgv ipaeeenpaf wnrqaaeald aakklqpiqk vaknlilflg 61 dglgvptvta trilkgqkng klgpetplam drfpylarsk tynvdrqvpd saatataylc 121 gvkanfqtig lsaaarfnqc nttrgnevis vmnrakqagk svgvvtttrv qhaspagtya 181 htvnrnwysd admpasarqe gcqdiatqli snmdidvilg ggrkymfpmg tpdpeypada 241 sqngirldgk nlvqewlakh qgawyvwnrt elmqasldqs vthlmglfep gdtkyeihrd 301 ptldpslmem teaalrllsr nprgfylfve ggridhghhe gvayqaltea vmfddaiera 361 gqltseedtl tlvtadhshv fsfggytlrg ssifglapsk aqdskaytsi lygngpgyvf 421 nsgvrpdvne sesgspdyqq qaavplsset hggedvavfa rgpqahlvhg vqeqsfvahv 481 mafaaclepy tacdlappac ttdaahpvaa slpllagtll llgasaap

[0025] Without being 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 some embodiments, an AP-based agent includes a mutation of the cysteine ​​(position 500 of SEQ ID NO:1). In some embodiments, the cysteine ​​is replaced with a glycine.

[0026] In various embodiments, the AP-based agent is bIAP II or a variant thereof. In some embodiments, bIAP II comprises the signal peptide and carboxy terminus of bIAP I. In some embodiments, bIAP II comprises an aspartic acid and position 248 (similar to bIAP IV). In some embodiments, bIAP II comprises the amino acid sequence of SEQ ID NO:2. bIAP II with 248D assignment - SEQ ID NO: 2. The signal peptide and sequence after 480 are from bIAP I. 1 mqgacvllll glhlqlslgl ipaeeenpaf wnrqaaqald vakklqpiqt aaknvilflg 61 dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121 gvkgnyrtig vsaaarynqc nttrgnevts vinrakkagk avgvvtttrv qhaspagaya 181 htvnrnwysd adlpadaqkn gcqdiaaqlv ynmdidvilg ggrmymfpeg tpdpeypdda 241 svngvrkdkq nlvqewqakh qgaqyvwnrt allqaaddss vthlmglfep admkynvqqd 301 htkdptlaem teaalqvlsr nprgfylfve ggridhghhd gkaymaltea imfdnaiaka 361 neltseldtl ilvtadhshv fsfggytlrg tsifglapgk aldsksytsi lygngpgyal 421 gggsrpdvng stseepsyrq qaavplaset hggedvavfa rgpqahlvhg vqeetfvahi 481 mafagcvepy tdcnlpapat atsipdaahl aasppplall agamllllap tly

[0027] In various embodiments, bIAP II comprises the amino acid sequence of SEQ ID NO:41. bIAP II with a stop codon and no leader sequence (SYN-020) (SEQ ID NO:41): LIPAEEENPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQC NTTRGNEVTSVINRAKKAGKAVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQKNGCQDIAAQLVYNMDIDVILGGGRMYMFPEGTPDPEYPDDASVNGVRKDKQNLVQEWQAKHQG AQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLAEMTEAALQVLSRNPRGFYLFVEGGRIDHGHHDGKAYMALTEAIMFDNAIAKANELTSELDTLILVTADHSHVFSFG GYTLRGTSIFGLAPGKALDSKSYTSILYGNGPGYALGGGSRPDVNGSTSEEPSYRQQAAVPLASETHGGEDVAVFARGPQAHLVHGVQEETFVAHIMAFAGCVEPYTDCNLPAPATATSIPD

[0028] In various embodiments, the AP-based agent is bIAP IV or a variant thereof. In one embodiment, bIAP IV comprises the amino acid sequence of SEQ ID NO:3. bIAP IV - Accession No. 3 1 mqwacvllll glwlqlsltf ipaeeedpaf wnrqaaqald vakklqpiqt aaknvilflg 61 dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121 gvkgnyktig vsaaarynqc nttsgnevts vmnrakkagk svgvvttsrv qhaspagaya 181 htvnrnwysd adlpadaqty gcqdiatqlv nnmdidvilg ggrmymfpeg tpdpeypydv 241 nqtgvrkdkr nlvqewqakh qgaqyvwnrt ellqaandps vthlmglfep admkynvqqd 301 ptkdptleem teaalqvlsr npqgfylfve ggridhghhe gkaymaltdt vmfdnaiaka 361 neltseldtl ilatadhshv fsfggytlrg tsifglapsk asdnksytsi lygngpgyvl 421 ggglrpdvnd sisedpsyrq qaavplsses hggedvavfa rgpqahlvhg vqeetfvahv 481 mafagcvepy tdcnlpapsg lsdaahlaas ppslallaga mllllapaly

[0029] Mammalian alkaline phosphatase is a glycosylphosphatidylinositol (GPI) anchored protein. It has a signal peptide and is translated into the secretory pathway. Once inside the endoplasmic reticulum (ER), the protein is glycosylated and folded. There are two disulfide bonds, as well as a single free cysteine ​​that is apparently not available from the surface. In the late ER, the carboxy terminus is removed, leaving the GPI anchor suspended. GPI anchoring is therefore a process that occurs at the carboxy terminus of alkaline phosphatase. The inclusion of a stop codon at the anchor site allows for the secretion of a biologically active protein (presumably a homodimer). Although there is no consensus sequence, the carboxy terminus contains three amino acids, designated omega, omega+1, and omega+2, which are followed by a short stretch of hydrophilic amino acids, followed by a stretch of hydrophobic amino acids. Without being bound by theory, it is believed that the hydrophobicity is important for embedding the carboxy terminus in the ER membrane. An enzymatic reaction then replaces the carboxy terminus with a GPI anchor.

[0030] In hPLAP, the GPI anchor binds to an aspartic acid of the sequence DAAH. Similarly, hIAP, bIAP II, and bIAP IV also have this DAAH conserved sequence that potentially functions as a GPI anchor site. Mutational studies of hPLAP indicate that prevention of GPI anchoring results in intracellular retention. In addition, mutations around the anchor site or in the hydrophobic domain either 1) prevent anchor binding, resulting in intracellular retention, or 2) do not prevent anchor binding. Without being bound by theory, it is believed that the hydrophobic domain functions as a signal for GPI anchor binding. Truncation or elimination of the hydrophobic domain results in secretion. Finally, a single mutation in the hydrophobic domain allows for the secretion of proteins with intact hydrophobic domains in hPLAP.

[0031] In various embodiments, the AP-based agents of the invention are GPI anchored to the host cell. For example, the AP-based agents can be GPI anchored to the plasma membrane of the host cell. In other embodiments, the AP-based agents of the invention are secreted proteins rather than anchored proteins. In some embodiments, the AP-based agents are not GPI anchored. In some embodiments, the AP-based agents lack a GPI anchor site. In some embodiments, the AP-based agents include a stop codon inserted immediately after the GPI anchor site. In some embodiments, the AP-based agents include a stop codon after the DAAH consensus site anchor (e.g., amino acid 503 of hIAP and bIAP IV, or amino acid 506 of bIAP II). hIAP with stop codon (SEQ ID NO: 4) 1 mqgpwvllll glrlqlslgv ipaeeenpaf wnrqaaeald aakklqpiqk vaknlilflg 61 dglgvptvta trilkgqkng klgpetplam drfpylarsk tynvdrqvpd saatataylc 121 gvkanfqtig lsaaarfnqc nttrgnevis vmnrakqagk svgvvtttrv qhaspagtya 181 htvnrnwysd admpasarqe gcqdiatqli snmdidvilg ggrkymfpmg tpdpeypada 241 sqngirldgk nlvqewlakh qgawyvwnrt elmqasldqs vthlmglfep gdtkyeihrd 301 ptldpslmem teaalrllsr nprgfylfve ggridhghhe gvayqaltea vmfddaiera 361 gqltseedtl tlvtadhshv fsfggytlrg ssifglapsk aqdskaytsi lygngpgyvf 421 nsgvrpdvne sesgspdyqq qaavplsset hggedvavfa rgpqahlvhg vqeqsfvahv 481 mafaaclepy tacdlappag ttd bIAP II with a stop codon (SEQ ID NO:5) 1 mqgacvllll glhlqlslgl ipaeeenpaf wnrqaaqald vakklqpiqt aaknvilflg 61 dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121 gvkgnyrtig vsaaarynqc nttrgnevts vinrakkagk avgvvtttrv qhaspagaya 181 htvnrnwysd adlpadaqkn gcqdiaaqlv ynmdidvilg ggrmymfpeg tpdpeypdda 241 svngvrkdkq nlvqewqakh qgaqyvwnrt allqaaddss vthlmglfep admkynvqqd 301 htkdptlaem teaalqvlsr nprgfylfve ggridhghhd gkaymaltea imfdnaiaka 361 neltseldtl ilvtadhshv fsfggytlrg tsifglapgk aldsksytsi lygngpgyal 421 gggsrpdvng stseepsyrq qaavplaset hggedvavfa rgpqahlvhg vqeetfvahi 481 mafagcvepy tdcnlpapat atsipd bIAP IV with a stop codon (SEQ ID NO:6) 1 mqwacvllll glwlqlsltf ipaeeedpaf wnrqaaqald vakklqpiqt aaknvilflg 61 dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121 gvkgnyktig vsaaarynqc nttsgnevts vmnrakkagk svgvvttsrv qhaspagaya 181 htvnrnwysd adlpadaqty gcqdiatqlv nnmdidvilg ggrmymfpeg tpdpeypydv 241 nqtgvrkdkr nlvqewqakh qgaqyvwnrt ellqaandps vthlmglfep admkynvqqd 301 ptkdptleem teaalqvlsr npqgfylfve ggridhghhe gkaymaltdt vmfdnaiaka 361 neltseldtl ilatadhshv fsfggytlrg tsifglapsk asdnksytsi lygngpgyvl 421 ggglrpdvnd sisedpsyrq qaavplsses hggedvavfa rgpqahlvhg vqeetfvahv 481 mafagcvepy tdcnlpapsg lsd

[0032] In one embodiment, the AP-based agent is bIAP IV, which includes a stop codon after amino acid 508 and mimics the secreted PLAP construct depicted below. bIAP IV (SEQ ID NO: 7) with a stop codon after amino acid 508 1 mqwacvllll glwlqlsltf ipaeeedpaf wnrqaaqald vakklqpiqt aaknvilflg 61 dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121 gvkgnyktig vsaaarynqc nttsgnevts vmnrakkagk svgvvttsrv qhaspagaya 181 htvnrnwysd adlpadaqty gcqdiatqlv nnmdidvilg ggrmymfpeg tpdpeypydv 241 nqtgvrkdkr nlvqewqakh qgaqyvwnrt ellqaandps vthlmglfep admkynvqqd 301 ptkdptleem teaalqvlsr npqgfylfve ggridhghhe gkaymaltdt vmfdnaiaka 361 neltseldtl ilatadhshv fsfggytlrg tsifglapsk asdnksytsi lygngpgyvl 421 ggglrpdvnd sisedpsyrq qaavplsses hggedvavfa rgpqahlvhg vqeetfvahv 481 mafagcvepy tdcnlpapsg lsdaahla

[0033] In various embodiments, the AP-based drug of the present invention is a fusion protein. In some embodiments, the AP-based drug comprises alkaline phosphatase fused to a protein domain that replaces the GPI anchor sequence. In some embodiments, the alkaline phosphatase is fused to a protein domain that facilitates protein folding, and / or protein purification, and / or protein dimerization, and / or protein stability. In various embodiments, the AP-based drug fusion protein has an extended serum half-life.

[0034] In certain embodiments, the alkaline phosphatase is fused to an immunoglobulin Fc domain and / or hinge region. In various 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 subclasses (IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In certain embodiments, the AP-based agent of the present invention comprises alkaline phosphatase fused to the hinge region and / or Fc domain of an IgG.

[0035] In various embodiments, the AP-based agents of the invention are fused to an IgG Fc domain that comprises one or more mutations. In some embodiments, the one or more mutations in the IgG Fc domain function to increase serum half-life and longevity. In some embodiments, the IgG Fc domain comprises one or more mutations at amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, US Public Health Service, National Institutes of Health, Washington, DC). In some embodiments, at least one of the amino acid substitutions is at amino acid residues 252, 254, 256, 309, 311, 433, or 434. In certain embodiments, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan, or threonine. In some embodiments, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In some embodiments, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In some embodiments, the amino acid substitution at amino acid residue 309 is a substitution with proline. In some embodiments, the amino acid substitution at amino acid residue 311 is a substitution with serine. In some embodiments, the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine, or glycine. In some embodiments, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In some embodiments, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In certain embodiments, the amino acid substitution at amino acid residue 389 is a substitution with a proline, a serine, or an asparagine.In some embodiments, the amino acid substitution at amino acid residue 433 is with arginine, serine, isoleucine, proline, or glutamine. In some embodiments, the amino acid substitution at amino acid residue 434 is with histidine, phenylalanine, or tyrosine.

[0036] In some embodiments, the IgG Fc domain comprises one or more mutations at amino acid residues 252, 254, 256, 433, 434, or 436. In one embodiment, the IgG Fc domain comprises a triple M252Y / S254T / T256E mutation or a YTE mutation. In another embodiment, the IgG Fc domain comprises a triple H433K / N434F / Y436H mutation or a KFH mutation. In a further embodiment, the IgG Fc domain comprises a combination of YTE and KFH mutations.

[0037] In some embodiments, the IgG Fc domain comprises one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435. Exemplary mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A. In some embodiments, the IgG Fc domain comprises M428L / N434S mutations or LS mutations. In another embodiment, the IgG Fc domain comprises T250Q / M428L mutations or QL mutations. In another embodiment, the IgG Fc domain comprises N434A mutations. In another embodiment, the IgG Fc domain comprises T307A / E380A / N434A mutations or AAA mutations. In another embodiment, the Fc domain of the IgG comprises I253A / H310A / H435A mutations or IHH mutations. In another embodiment, the Fc domain of the IgG comprises H433K / N434F mutations. In another embodiment, the Fc domain of the IgG region comprises a combination of M252Y / S254T / T256E and H433K / N434F mutations.

[0038] Exemplary mutations in the Fc domain of IgG are described, for example, in Robbie, et al., Antimicrobial Agents and Chemotherapy (2013), 57(12):6147-6153; Dall'Acqua et al., JBC (2006), 281(33):23514-24; Dall'Acqua et al., Journal of Immunology (2002), 169:5171-80; Ko et al. Nature (2014) 514:642-645; Grevys et al Journal of Immunology. (2015), 194(11):5497-508; and U.S. Patent No. 7,083,784, the contents of all of which are incorporated herein by reference.

[0039] In various embodiments, one or more mutations in the Fc domain of an IgG increase affinity for the neonatal Fc receptor (FcRn), hi some embodiments, one or more mutations in the Fc domain of an IgG increase affinity for FcRn at a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5.

[0040] In various embodiments, the alkaline phosphatase is condensed to one or more of PEG, XTENylation (e.g., rPEG), polysialic acid (POLYXEN), albumin, elastin-like protein, elastin-like protein (ELP), PAS, HAP, GLK, CTP, and transferrin. In various embodiments, the alkaline phosphatase is fused to one or more of the agents described in BioDrugs (2015) 29:215-239, the entire contents of which are incorporated herein by reference.

[0041] In some embodiments, alkaline phosphatase is fused to a protein domain (e.g., an immunoglobulin Fc domain) via a linker to a GPI anchor site. For example, alkaline phosphatase can be fused to a protein domain via an aspartic acid of a GPI anchor sequence. The present invention contemplates the use of various linker sequences. In various embodiments, the linker can be derived from a naturally occurring multi-domain protein, or is an empirical linker, for example, as described in Chichili et al., (2013), Protein Sci. 22(2): 153-167; Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369, the contents of which are all incorporated herein by reference. In some embodiments, the linker may be designed using linker design databases and computer programs, such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto et al., (2000), Protein Eng. 13(5): 309-312, all of which are incorporated herein by reference. In various embodiments, the linker may be functional. For example, without limitation, the linker may function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve the biological activity of the AP-based agent of the present invention. In another example, the linker may function to target the AP-based agent to a specific cell type or location.

[0042] In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids in length. For example, the linker can be less than about 100, less than about 95, less than about 90, less than about 85, less than about 80, less than about 75, less than about 70, less than about 65, less than about 60, less than about 55, less than about 50, less than about 45, less than about 40, less than about 35, less than about 30, less than about 25, less than about 20, less than about 19, less than about 18, less than about 17, less than about 16, less than about 15, less than about 14, less than about 13, less than about 12, less than about 11, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, or less than about 2 amino acids in length. In some embodiments, the linker is flexible. In other embodiments, the linker is rigid.

[0043] In various embodiments, the linker is substantially composed of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 19). Additional exemplary linkers include the sequences LE, GGGGS (SEQ ID NO: 20), (GGGGS) n (n = 2 to 4) (SEQ ID NOs: 21 to 23), (Gly) 8 (SEQ ID NO: 24), (Gly) 6 (SEQ ID NO: 25), (EAAAK) n (n = 1 to 3) (SEQ ID NOs: 26 to 28), A(EAAAK) n A(n=2 to 5)(SEQ ID NO:29 to 32), AEAAAKEAAAKA(SEQ ID NO:30), A(EAAAK) 4 ALEA(EAAAK) 4 A (SEQ ID NO: 33), PAPAP (SEQ ID NO: 34), KESGSVSSEQLAQFRSLD (SEQ ID NO: 35), EGKSSGSGSESKST (SEQ ID NO: 36), GSAGSAAGSGEF (SEQ ID NO: 37), and (XP) nwhere X represents any amino acid, e.g., Ala, Lys, or Glu. In various embodiments, the linker is GGS.

[0044] In some embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2). The hinge region found in antibodies of the IgG, IgA, IgD, and IgE classes acts as a flexible spacer, allowing the Fab portions to move freely in space. In contrast to constant regions, hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231 and is freely mobile, allowing the Fab fragment to rotate around the axis of symmetry and move within a spherical range around the first two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix stabilized by other inter-heavy chain disulfide bridges. These properties limit the mobility of the IgG2 molecule. IgG3 differs from the other subclasses by a unique extended hinge region (approximately four times longer than the IgG1 hinge), which contains 62 amino acids (including 21 prolines and 11 cysteines) to form a non-mobile polyproline double helix. In IgG3, the Fab fragment is relatively far from the Fc fragment, giving the molecule greater mobility. The extended hinge of IgG3 is also responsible for its higher molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between that of IgG1 and IgG2. It has been reported that the flexibility of the hinge region decreases in the following order: IgG3>IgG1>IgG4>IgG2.

[0045] Crystallographic studies have shown that the immunoglobulin hinge region can be functionally further subdivided into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region is C H1 The upper hinge region includes amino acids from the carboxy terminus of the C to the first residue that restricts movement in the hinge, typically to the first cysteine ​​residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region includes amino acids from the carboxy terminus of the C H2 Linked to the amino terminus of the domain, C H2 The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys, which when dimerized by disulfide bond formation results in a cyclic octapeptide that is believed to act as a pivot point, thereby conferring flexibility. In various embodiments, the linkers of the invention comprise one, two, or three of the upper hinge region, core region, and lower hinge region of any antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (IgG1, IgG2, IgG3, and IgG4, as well as IgA1 and IgA2)). The hinge region can also contain one or more glycosylation sites, which include a number of structurally unique types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region, conferring resistance to intestinal proteases to the hinge region polypeptide, which is considered an advantageous property for secretory immunoglobulins. In various embodiments, the linkers of the invention contain one or more glycosylation sites.

[0046] In some embodiments, the linker is a synthetic linker, such as PEG.

[0047] Exemplary FC fusion constructs of the invention include the following:

[0048] bIAP II with Fc fusion (SEQ ID NO: 8) - Fc domain is underlined

[0049] [Table 1]

[0050] bIAP IV (SEQ ID NO: 9) with Fc fusion - Fc domain is underlined

[0051] [Table 2]

[0052] The hIAP-Fc domain with Fc fusion is underlined: SEQ ID NO: 18

[0053] [Table 3]

[0054] In various embodiments, the linker may be replaced by any other linker disclosed herein.

[0055] Saccharomyces alkaline phosphatase Pho8 is produced as an inactive zymogen. It is a transmembrane protein that is not GPI-anchored but has an amino terminus that extends from the lysosome into the cytoplasm. Within the lysosome, the enzyme PEP4 cleaves the carboxy terminus, activating the enzyme. Without being bound by theory, it is believed that mammalian alkaline phosphatases may also be produced as inactive zymogens. This is because alkaline phosphatases may dephosphorylate ATP, thereby allowing activity within the ER to deplete the ER's main energy source. Without being bound by theory, it is believed that the inhibitory function is located at the carboxy terminus that is released by the addition of a GPI anchor. Alternatively, other activities, such as folding or metal (Zn or Mg) inclusion, may control activity.

[0056] In various embodiments, the AP-based agent of the present invention is a zymogen. In some embodiments, the activity of the zymogen is inhibited by the carboxy terminus. In some embodiments, protease removal of the carboxy terminus reactivates the alkaline phosphatase enzyme activity. In certain embodiments, the zymogen is secreted more efficiently than the enzyme without the carboxy terminus.

[0057] In some embodiments, the native carboxy terminus of alkaline phosphatase is replaced with the similar sequence of hPLAP in the generation of the zymogen. In some embodiments, mutations are made in the hydrophobic carboxy tail to facilitate protein secretion without truncating the carboxy terminus. In an exemplary embodiment, a single point mutation, such as, for example, a substitution of leucine with arginine, is made in the hydrophobic carboxy terminus (e.g., changing allpllagtl to, for example, allplragtl), resulting in secretion of the enzyme without removing the carboxy terminus.

[0058] In some embodiments, the AP-based drug is modified to contain a specific enzyme cleavage site that allows for subsequent removal of the carboxy terminus. In some embodiments, the AP-based drug contains a protease cleavage site. Exemplary protease cleavage sites include, but are not limited to, cleavage sites recognized by furin, rhinovirus 16 3C protease, factor Xa protease, trpysin, chymotrypsin, elastase, pepsin, papain subtilisin, thermolysin, V-8 protease, submaxillaris protease, clostripain, thrombin, collagenase, and any other endoprotease. In alternative embodiments, the AP-based drug contains a cleavage site recognized by digestive enzymes present in the GI tract. In such embodiments, the AP-based drug may be administered as a prodrug that is subsequently activated in the GI tract.

[0059] In an exemplary embodiment, the zymogen is a bIAP IV zymogen having the following sequence:

[0060] bIAP IV with hPLAP carboxy terminus, non-processed secretion mutation, and RV3C cleavage site (...LEVLFQGP...): SEQ ID NO: 10

[0061] [Table 4]

[0062] bIAP IV with hPLAP carboxy terminus, unprocessed secretion mutation, and FXa cleavage site (...IEGR...): SEQ ID NO: 11

[0063] [Table 5]

[0064] In various embodiments, the AP-based agents of the invention are efficiently expressed and secreted in the host cell. In some embodiments, the AP-based agents of the invention are efficiently transcribed in the host cell. In another embodiment, the AP-based agents exhibit enhanced RNA stability and / or transport in the host cell. In another embodiment, the AP-based agents are efficiently translated in the host cell. In further embodiments, the AP-based agents fold and / or translocate efficiently through the ER, pre-golgi, and golgi. In another embodiment, the AP-based agents exhibit enhanced protein stability.

[0065] In various embodiments, the AP-based agent is GPI anchored to the plasma membrane of the host cell. In other embodiments, the AP-based agent of the present invention is secreted from the host cell. In such embodiments, the AP-based agent can include a protease cleavage site immediately upstream of the GPI anchor site. Exemplary protease cleavage sites have been described. In one embodiment, the protease cleavage site is a furin cleavage site. In another embodiment, the AP-based agent can include a cleavage site immediately upstream of the GI anchor site that is recognized by digestive enzymes of the GI tract. In these embodiments, the AP-based agent is anchored to the ER and released to the late golgi and secreted.

[0066] In various embodiments, the AP-based agent is efficiently expressed in the host cell. In some embodiments, the Kozak sequence of the DNA construct encoding the AP-based agent is optimized. The Kozak sequence is a nucleotide sequence flanking the ATG start codon that instructs the liposome to start translation. Although there is flexibility in the design of the Kozak sequence, one canonical sequence is GCCGCCACCATGG (SEQ ID NO: 38). The purine at position -3 and the G at position +4 are the most important bases for translation initiation. In hIAP, bIAP II, and bIAP IV, the second amino acid, the amino acid immediately following the initiator methionine, is glutamine. All codons for glutamine have a C at position 1. Thus, the Kozak sequences all have the ATGC (SEQ ID NO: 39) sequence. Thus, in various embodiments, the ATGC (SEQ ID NO: 39) sequence is changed to ATGG (SEQ ID NO: 40). This is accomplished by changing the second amino acid to glycine, alanine, valine, aspartic acid, or glutamic acid, all of which have a G at position 1 in their codons. These amino acids may be compatible with 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.

[0067] In various embodiments, the signal peptide of an AP-based agent can be deleted and / or replaced, for example, the signal peptide can be deleted, mutated, and / or replaced (e.g., by another signal peptide) to ensure protein expression.

[0068] In some embodiments, the DNA constructs encoding the AP-based agents of the invention include non-translated DNA sequences. Such sequences include introns, which may be heterologous to the IAP protein, or may be native to the IAP protein, including the native first and / or second introns, and / or the native 3'UTR. Without being bound by theory, it is believed that the inclusion of these sequences stabilizes the mRNA and enhances protein expression. Thus, in various embodiments, the DNA constructs encoding the AP-based agents of the invention include the 5'UTR and / or the 3'UTR.

[0069] Presented below is the DNA sequence of an exemplary IAP, including the first intron and 3'UTR.

[0070] hIAP-SEQ ID NO: 12 with native first intron (shown in bold and underlined)

[0071] [Table 6]

[0072] hIAP-SEQ ID NO: 13 with native 3'UTR (shown in bold and underlined)

[0073] [Table 7]

[0074] hIAP IV with the first intron of bIAP I (shown in bold and underlined) - SEQ ID NO: 14

[0075] [Table 8]

[0076] hIAP IV with the 3'UTR of bIAP I (shown in bold and underlined) - SEQ ID NO: 15

[0077] [Table 9]

[0078] In various embodiments, the present invention contemplates the use of bacterial alkaline phosphatase. In some embodiments, the AP-based agents of the present invention are derived from Bacillus subtilis. Bacillus subtilis is a Gram-positive bacterium found in soil and the human gastrointestinal tract. Bacillus subtilis secretes high levels of correctly folded proteins into the environment and into the human GI tract. Without being bound by theory, it is believed that Bacillus subtilis that secrete proteins into the GI tract may be resistant to digestion by common gastrointestinal proteases. Bacillus subtilis expresses high levels of the alkaline phosphatase multigene family. Of these isozymes, alkaline phosphatase IV is responsible for the majority of the total alkaline phosphatase expression and activity in B. subtilis. In some embodiments, the AP-based agents of the present invention are derived from Bacillus licheniformis. In some embodiments, the AP-based agents of the present invention are derived from Escherichia coli.

[0079] Thus, in an exemplary embodiment, the AP-based agents of the invention are derived from Bacillus subtilis alkaline phosphatase IV. In certain embodiments, the cellular alkaline phosphatase may have the following nucleotide and amino acid sequences:

[0080] Bacillus subtilis JH642 alkaline phosphatase IV mature protein nucleotide sequence - SEQ ID NO: 16

[0081] [Table 10]

[0082] Bacillus subtilis JH642 Alkaline Phosphatase IV Mature Protein Amino Acids - SEQ ID NO: 17

[0083] [Table 11]

[0084] In some embodiments, the AP-based agent comprises a bacterial alkaline phosphatase having one or more mutations that alter its catalytic activity. In some embodiments, the bacterial alkaline phosphatase comprises one or more mutations that make its catalytic activity similar to or higher than that of a mammalian alkaline phosphatase. In some embodiments, the bacterial alkaline phosphatase comprises one or more mutations that alter its dephosphorylation profile. In certain embodiments, the bacterial alkaline phosphatase of the present invention exhibits a dephosphorylation profile similar to that of a mammalian alkaline phosphatase. In some embodiments, the bacterial alkaline phosphatase comprises one or more mutations that alter its activity at high pH. In certain embodiments, the bacterial alkaline phosphatase of the present invention exhibits an activity at high pH similar to that of a mammalian alkaline phosphatase. In some embodiments, the bacterial alkaline phosphatase comprises one or more mutations that alter its metal requirement. In certain embodiments, the bacterial alkaline phosphatase of the present invention exhibits a metal (e.g., Mg) requirement similar to that of a mammalian alkaline phosphatase.

[0085] For example, in certain embodiments, an AP-based agent of the invention is derived from Bacillus subtilis JH642 alkaline phosphatase IV and has one or more mutations at positions 101, 328, A330, and 374. For example, an AP-based agent can include one or more of the following mutations: D101A, W328H, A330N, and G374C.

[0086] In various embodiments, the AP-based agents of the invention have at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77% or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity to the sequence.

[0087] In some embodiments, the AP-based agents of the invention are at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100%, or about 101%, or about 102%, or about 103%, or about 104%, or about 105%, or about 106%, or about 107%, or about 108%, or about 109%, or about 110%, or about 111%, or about 112%, or about 113%, or about 114%, or about 115%, or about 116%, or about 117%, or about 118%, or about 119%, or about 120%, or about 121%, or about 122%, or about 123%, or about 124%, or about 125%, or about 126%, or about 127%, or about 128%, or about 129%, or about 130%, or about 131%, or about 132, or about 133, or about 134, or %, 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 amino acid sequence.

[0088] In various embodiments, the AP-based agents of the present invention can include an amino acid sequence having one or more amino acid mutations relative to any of the protein sequences described herein. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0089] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0090] "Conservative substitutions" can be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0091] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the replacement of Asp with Glu causes the modified polypeptide to retain one negative charge. In addition, glycine and proline can be substituted for each other based on their ability to break alpha helices.

[0092] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[0093] In various embodiments, substitutions can also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-forms 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, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and common amino acid analogs).

[0094] Mutations can also be made to the nucleotide sequence of alkaline phosphatase with reference to the genetic code, taking into account codon degeneracy. In various embodiments, DNA constructs encoding AP-based agents are codon optimized for protein expression in host cells.

[0095] Mutations can be made to the AP-based drugs of the invention to select drugs with desired properties. For example, mutations can be made to generate AP-based drugs with enhanced enzymatic activity or protein stability. In various embodiments, directed evolution can be used to generate AP-based drugs of the invention. For example, error-prone PCR and DNA shuffling can be used to identify mutations in bacterial alkaline phosphatase that confer enhanced activity.

[0096] In various embodiments, the AP-based agents of the invention have desirable properties including, for example, high specific activity. In various embodiments, the alkaline phosphatases of the invention have a specific activity of at least about 100 U / mg to about 20,000 U / mg. In various embodiments, the alkaline phosphatases of the invention have a specific activity of at least about 100 U / mg, about 200 U / mg, about 300 U / mg, about 400 U / mg, about 500 U / mg, about 600 U / mg, about 700 U / mg, about 800 U / mg, about 900 U / mg, about 1,000 U / mg, about 2,000 U / mg, about 3,000 U / mg, about 4,000 U / mg, about 5,000 U / mg, about 6,000 U / mg, or about 7,000 U / mg. g, and has a specific activity of about 7,000 U / mg, about 8,000 U / mg, about 9,000 U / mg, about 10,000 U / mg, about 11,000 U / mg, about 12,000 U / mg, about 13,000 U / mg, about 14,000 U / mg, about 15,000 U / mg, about 16,000 U / mg, about 17,000 U / mg, about 18,000 U / mg, about 19,000 U / mg, or about 20,000 U / mg.

[0097] In various embodiments, the AP-based agent of the present invention is stable and / or active in the GI tract, for example, in 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, the alkaline phosphatase is stable in the large intestine, optionally selected from one or more of the transverse colon, descending colon, ascending colon, sigmoid colon, and cecum. In certain embodiments, the alkaline phosphatase is stable in the small intestine, optionally selected from one or more of the duodenum, jejunum, and ileum. In some embodiments, the alkaline phosphatase is resistant to proteases in the GI tract, including, for example, the small intestine. In some embodiments, the alkaline phosphatase is substantially active at a pH of about 5.0 or higher. For example, the alkaline phosphatase may be substantially active (including, for example, via a formulation described herein) 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. In some embodiments, stable refers to an enzyme that has a sufficiently long half-life and maintains sufficient activity for therapeutic efficacy.

[0098] In various embodiments, the AP-based agents of the present invention are stable in chyme.

[0099] In some embodiments, the AP-based agents described herein include derivatives that are modified, i.e., any type of molecule is covalently attached to alkaline phosphatase such that the covalent attachment does not prevent the activity of the enzyme. For example, without limitation, derivatives include alkaline phosphatase modified by, among others, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, derivatives can contain one or more non-classical amino acids. In various embodiments, the AP-based agents are glycosylated to ensure correct protein folding.

[0100] In yet other embodiments, the AP-based agents of the present invention can be modified by the addition of effector moieties, such as chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties, such as streptavidin, avidin, biotin, cytotoxins, cytotoxic agents, and radioactive materials.

[0101] The AP-based drugs described herein may have a sufficiently basic functional group that can react with inorganic or organic acids, or a carboxyl group that can react with inorganic or organic bases to form pharmaceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids and are well known in the art. Such salts are listed, for example, in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P.H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are incorporated herein by reference in their entirety.

[0102] Pharmaceutically acceptable salts include, by way of non-limiting example, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenyl acetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, benzoic acid salts ... These include benzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, tetraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate.

[0103] 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, alkali metal hydroxides, such as sodium, potassium, and lithium; alkaline earth metal hydroxides, such as calcium and magnesium; other metal hydroxides, such as aluminum and zinc; ammonia and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines, 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-(hydroxyl-lower alkyl)-amines, 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.

[0104] In some embodiments, the compositions described herein are in the form of a pharma- ceutically acceptable salt.

[0105] Furthermore, any of the AP-based agents described herein may be administered to a subject as a component of a composition that includes a pharma- ceutically acceptable carrier or vehicle. Such compositions may optionally contain an appropriate amount of a pharma- ceutically acceptable excipient to provide a precise dosage form.

[0106] The pharmaceutical excipients may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The pharmaceutical excipients may be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary agents, stabilizers, thickening agents, lubricants, and coloring agents may be used. In one embodiment, the pharma-ceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be specifically used as liquid excipients for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, cellulose, hypromellose, lactose, sucrose, corn, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, povidone, crospovidone, water, ethanol, etc. Any agent described herein may 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.

[0107] If necessary, the AP-based drug and / or pharmaceutical composition (and / or additional therapeutic agent) can include a solubilizing agent. The drug can also be delivered by a suitable vehicle or delivery device. The composition for administration can optionally include a local anesthetic, such as lignocaine, to ease pain at the injection site. The combination therapy outlined herein can be delivered simultaneously in a single delivery vehicle or delivery device.

[0108] How to make IAP The IAPs of the present invention are produced using standard molecular biology techniques. For example, nucleic acid compositions encoding the IAPs of the present invention are also provided, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acids and / or expression vector compositions. As will be appreciated by those skilled in the art, the protein sequences depicted herein may be encoded by any number of possible nucleic acid sequences due to the degeneracy of the genetic code.

[0109] As known in the art, the nucleic acid encoding the components of the present invention is incorporated into an expression vector, which is known in the art and can be used to produce the IAP composition of the present invention depending on the host cell. Generally, the nucleic acid is operably linked to a number of regulatory elements (promoter, transcription origin, selectable marker, ribosome binding site, inducer, etc.). The expression vector can be an extrachromosomal vector or an integrating vector.

[0110] The nucleic acids and / or expression vectors of the invention are then transformed into a number of different types of host cells, including mammalian cells, bacterial cells, yeast cells, insect cells, and / or fungal cells, as are well known in the art, and find use in many embodiments with mammalian cells (e.g., CHO cells).

[0111] The IAPs of the invention are produced by culturing host cells containing the expression vectors, as is well known in the art. Once produced, traditional purification steps are performed.

[0112] Preparations containing AP-based drugs The present invention provides the described AP-based medicaments and / or pharmaceutical compositions (and / or additional therapeutic agents) in various formulations. Any AP-based medicament and / or pharmaceutical composition (and / or additional therapeutic agent) described herein can be in the form of a tablet, pill, pellet, capsule, capsule containing a liquid, capsule containing multiparticulates, powder, liquid, emulsion, drops, suppository, emulsion, aerosol, spray, suspension, delayed release formulation, sustained release formulation, controlled release formulation, or any other form suitable for use.

[0113] Formulations containing AP-based drugs and / or pharmaceutical compositions (and / or additional therapeutic agents) can be conveniently presented in unit dosage form. For example, dosage forms can be prepared by a process that includes the step of bringing the therapeutic agent into association with a carrier that constitutes one or more accessory ingredients. For example, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired dosage form (e.g., wet or dry granulation, powder blending, etc., followed by compression molding).

[0114] In one embodiment, the IAP (and / or additional therapeutic agent) described herein is formulated into a composition compatible with a mode of administration described herein.

[0115] In some embodiments, administration of the IAP and / or pharmaceutical composition (and / or additional therapeutic agent) is any one of oral, intravenous, and parenteral. For example, routes of administration include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical (e.g., ear, nose, eye, or skin).

[0116] In some embodiments, the IAP and / or pharmaceutical compositions (and / or additional therapeutic agents) described herein are formulated as compositions adapted for oral administration. Compositions for oral delivery may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, sprinkles, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more agents, such as sweeteners, for example, fructose, aspartame, or saccharin; flavoring agents, such as peppermint, wintergreen oil, or cherry; coloring agents; and preservatives, to provide a medicamentously palatable preparation. Additionally, in tablet or pill form, the compositions may be coated to retard disintegration and provide a sustained action over an extended period of time. Selectively permeable membranes surrounding any alkaline phosphatase (and / or additional therapeutic agent) inducing osmotically active agents described herein are also suitable for orally administered compositions. In the latter platform, fluid from the capsule's surrounding environment is imbibed by the derivative compound, which expands and pushes the drug or drug composition out of the opening. These delivery platforms can provide an essentially zero-order delivery profile, as opposed to the spiked profiles of immediate release formulations. Time-delay materials such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can include standard excipients, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, ethacrylic acid and its derivative polymers, and magnesium carbonate. In one embodiment, the excipients are of pharmaceutical grade. Suspensions may contain suspending agents, in addition to the active compound, such as ethoxylated isostearyl alcohol, polyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and the like, and mixtures thereof.

[0117] In various embodiments, the AP-based agent and / or pharmaceutical composition (and / or additional therapeutic agent) is formulated as a solid dosage form, such as a tablet, dispersible powder, granules, and capsule. In one embodiment, the AP-based agent and / or pharmaceutical composition (and / or additional therapeutic agent) is formulated as a capsule. In another embodiment, the AP-based agent and / or pharmaceutical composition (and / or additional therapeutic agent) is formulated as a tablet. In yet another embodiment, the AP-based agent and / or pharmaceutical composition (and / or additional therapeutic agent) is formulated as a softgel capsule. In a further embodiment, the AP-based agent and / or pharmaceutical composition (and / or additional therapeutic agent) is formulated as a gelatin capsule.

[0118] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarterial injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, etc. They can also be prepared in the form of sterile solid compositions (e.g., lyophilized compositions), which can be dissolved or suspended in a sterile injectable medium immediately before use. For example, they may contain suspending agents or dispersing agents.

[0119] In various embodiments, the formulation of an AP-based drug, such as an IAP, can additionally include a pharma- ceutically acceptable carrier or excipient. As will be appreciated by those skilled in the art, the formulation can be in any suitable form suitable for the desired use and route of administration.

[0120] In some dosage forms, the agents described herein are mixed with at least one inert pharma- ceutically acceptable excipient or carrier, such as, for example, sodium citrate, dicalcium phosphate, and / or a) fillers or extenders, such as, for example, starch, lactose, sucrose, glucose, mannitol, silicic acid, microcrystalline cellulose, and Bakers Special Sugar; b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylcellulose (HPC), and hydroxymethylcellulose; c) humectants, such as, for example, glycerol; d) disintegrants, such as, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, cross-linked polymers, such as, for example, crospovidone (cross-linked polyvinylpyrrolidone), croscarmellose sodium (cross-linked sodium carboxymethylcellulose), sodium starch glycolate, sodium glycerol, sodium sorbitol ... glycolate), e) dissolution retarders, such as paraffin, f) absorption enhancers, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, glyceryl behenate, and mixtures of such excipients. Those skilled in the art will recognize that a particular excipient may have more than one function 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.

[0121] The formulation may additionally comprise a surfactant.Surfactants suitable for use in the present invention include, but are not limited to, any pharma- ceutically acceptable non-toxic surfactant.Surfactant classes suitable for use in the present composition include, but are not limited to, polyethoxylated fatty acids, PEG-fatty acid diesters, PEG-fatty acid mono- and diester mixtures, polyethylene glycol glycerol fatty acid esters, alcohol-oil transesterification products, polyglycerinated fatty acids, propylene glycol fatty acid esters, mixtures of propylene glycol esters-glycerol esters, mono- and diglycerides, sterols and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar esters, polyethylene glycol alkyl phenols, polyoxyethylene-polyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, ionic surfactants, and mixtures thereof. In some embodiments, the compositions of the present invention may contain one or more surfactants, including but not limited to sodium lauryl sulfate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and triethyl citrate.

[0122] The formulation may contain pharma- ceutically acceptable plasticizers to obtain desired mechanical properties, such as flexibility and hardness, including, but not limited to, triacetin, citrate esters, triethyl citrate, phthalate esters, dibutyl sebacate, cetyl alcohol, polyethylene glycol, polysorbates, or other plasticizers.

[0123] The formulation may also include one or more solvents for application. More common solvents that may be used for application, for example delayed release coating compositions, include isopropyl alcohol, acetone, methylene chloride, and the like.

[0124] The formulation can also include one or more alkaline substances.The alkaline substances suitable for use in the compositions of the present invention include, but are not limited to, sodium, potassium, calcium, magnesium, and aluminum salts of acids such as phosphate, carbonate, citric acid, and other aluminum / magnesium compounds.In addition, the alkaline substance can be selected from antacid substances such as aluminum hydroxide, calcium hydroxide, magnesium hydroxide, and magnesium oxide.

[0125] In various embodiments, the formulation can additionally include magnesium and / or zinc. Including magnesium and / or zinc in the formulation can promote protein folding (e.g., dimer formation) and biological activity of AP-based agents. In some embodiments, the formulation can include magnesium at a concentration of about 1 μM to greater than 500 mM (e.g., about 1 μM to greater than 5 mM), including all ranges and values ​​therebetween. In some embodiments, the formulation can include zinc at a concentration of about 1 μM to greater than 100 mM (e.g., about 1 μM to greater than 1 mM), including all ranges and values ​​therebetween. In various embodiments, the formulations of the present invention are substantially free of metal chelators.

[0126] In various embodiments, the pH of the formulation ensures that the AP-based agent is correctly folded (e.g., dimerized) and biologically active. In some embodiments, the formulation is maintained at a pH such that the amino acids that coordinate the magnesium and / or zinc bonds in the AP-based agent are not protonated. Protonation of such coordinated amino acids can result in loss of metal ions and biological activity, as well as dimer dissociation. In various 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.

[0127] Besides inert diluents, the oral compositions can also include adjuvants such as sweetening, flavoring, and perfuming agents.

[0128] In various embodiments, the AP-based agents and / or pharmaceutical compositions (and / or additional therapeutic agents) are formulated for systemic or local delivery. In certain embodiments, administration is systemic. In other embodiments, it may be desirable to administer locally to the area in need of treatment.

[0129] Various methods can be used to formulate and / or deliver the agents described herein to a desired location. For example, the intestinal alkaline phosphatase and / or pharmaceutical composition (and / or additional therapeutic agent) described herein can be formulated for delivery to the gastrointestinal tract. The gastrointestinal tract includes organs of the digestive system such as the mouth, esophagus, stomach, duodenum, small intestine, large intestine, and rectum, including all of these subdivisions (e.g., the small intestine may include the duodenum, jejunum, and ileum, and the large intestine may include the transverse colon, descending colon, ascending colon, sigmoid colon, and cecum). For example, the alkaline phosphatase and / or pharmaceutical composition (and / or additional therapeutic agent) described herein can be formulated for delivery to one or more of the stomach, small intestine, large intestine, and rectum, including all of these subdivisions (e.g., the duodenum, jejunum, and ileum, the transverse colon, descending colon, ascending colon, sigmoid colon, and cecum). In some embodiments, the compositions described herein may be formulated for delivery to the upper or lower GI tract. In certain embodiments, the alkaline phosphatase and / or pharmaceutical composition (and / or additional therapeutic agent) may be administered to a subject, for example, by direct or indirect contact with mucosal tissue of the gastrointestinal tract.

[0130] In various embodiments, administration of the AP-based agent and / or pharmaceutical composition (and / or additional therapeutic agent) is into the GI tract via, for example, oral delivery, a nasogastral tube, intestinal intubation (e.g., an enteral or gastric tube, such as a jejunal or gastrojejunal tube), direct injection (e.g., duodenal injection), endoscopy, colonoscopy, or enema.

[0131] Administration and Dosage As those skilled in the art will understand, the actual dose of IAP administered according to the present invention will vary depending on the specific compound, the specific dosage form, and the mode of administration.Many factors that can modify the action of IAP (e.g., body weight, sex, diet, administration time, administration route, excretion rate, subject's condition, drug combination, genetic tendency, and reaction sensitivity) can be considered by those skilled in the art.Administration can be performed continuously, or can be performed in one or more separate doses within the maximum tolerated dose.The administration rate in a given condition can be confirmed by those skilled in the art using conventional dosage administration tests.

[0132] The individual doses of AP-based drugs, e.g., IAP, are, 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 to about 100 mg, about 0. It may be administered in a unit dosage form (e.g., a tablet or capsule) containing 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 include about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 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 64 mg 3mg, 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 30mg , 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 4 8mg, about 49mg, about 50mg, about 51mg, about 52mg, about 53mg, about 54mg, about 55mg, about 56mg, about 57mg, about 58mg, about 59mg, about 60mg, about 61mg, about 62mg, about 63mg, about 64mg, about 65mg, about 66mg, about 67mg, about 68mg, about 69mg, about 70mg, about 71mg, about 72mg, about 73mg, about 74mg, about 75mg, about 76mg, about 77mg, about 78mg, about 79mg, about 80mg, about 81mg, about 82mg, about 8 The dosage form may be about 3 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 of the AP-based drug (including all values ​​and ranges therebetween).

[0133] In one embodiment, an AP-based agent, such as an IAP, 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 80 mg per day, The dose is about 0.1 mg to about 75 mg per day, about 0.1 mg to about 70 mg per day, about 0.1 mg to about 65 mg per day, about 0.1 mg to about 60 mg per day, about 0.1 mg to about 55 mg per day, about 0.1 mg to about 50 mg per day, about 0.1 mg to about 45 mg per day, about 0.1 mg to about 40 mg per day, about 0.1 mg to about 35 mg per day, about 0.1 mg to about 30 mg per day, about 0.1 mg to about 25 mg per day, about 0.1 mg to about 20 mg per day, about 0.1 mg to about 15 mg per day, about 0.1 mg to about 10 mg per day, about 0.1 mg to about 5 mg per day, about 0.1 mg to about 3 mg per day, about 0.1 mg to about 1 mg per day, or about 5 mg to about 80 mg per day.In various embodiments, the AP-based agent is about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 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 6 mg, about 12mg, 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 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 , about 47mg, about 48mg, about 49mg, about 50mg, about 51mg, about 52mg, about 53mg, about 54mg, about 55mg, about 56mg, about 57mg, about 58mg, about 59mg, about 60mg, about 61mg, about 62mg, about 63mg, about 64mg, about 65mg, about 66mg, about 67mg, about 68mg, about 69mg, about 70mg, about 71mg, about 72mg, about 73mg, about 74mg, about 75mg, about 76mg, about 77mg, about 78mg, about 79mg, about 80mg, about 81mg, and / or 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).

[0134] In some embodiments, a suitable dosage of an AP-based agent 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, 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 1 ...00 mg / kg of the subject's body weight, 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 100 mg / kg of the subject's body weight, from about 0.01 mg / kg to about 100 The range is from about 0.01 mg / kg to about 40 mg / kg of the subject's body weight, from about 0.01 mg / kg to about 30 mg / kg of the subject's body weight, from about 0.01 mg / kg to about 20 mg / kg of the subject's body weight, from about 0.01 mg / kg to about 10 mg / kg of the subject's body weight, for example, about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, ... g / kg, approximately 0.1mg / kg, approximately 0.2mg / kg, approximately 0.3mg / kg, approximately 0.4mg / kg, approximately 0.5mg / kg, approximately 0.6mg / kg, approximately 0.7mg / kg, approximately 0.8mg / kg, approximately 0.9mg / kg, approximately 1m g / kg, approximately 1.1mg / kg, approximately 1.2mg / kg, approximately 1.3mg / kg, approximately 1.4mg / kg, approximately 1.5mg / kg, approximately 1.6mg / kg, approximately 1.7mg / kg, approximately 1.8mg / kg, 1.9mg / kg, approximately 2mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, 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 other embodiments, a suitable dosage of an AP-based agent is in the range of about 0.01 mg / kg to about 10 mg / kg of body weight, in the range of about 0.01 mg / kg to about 9 mg / kg of body weight, in the range of about 0.01 mg / kg to about 8 mg / kg of body weight, in the range of about 0.01 mg / kg to about 7 mg / kg of body weight, in the range of about 0.01 mg / kg to about 6 mg / kg of body weight, in the range of about 0.05 mg / kg to about 5 mg / kg of body weight, in the range of about 0.05 mg / kg to about 4 mg / kg of body weight, in the range of about 0.05 mg / kg to about 3 mg / kg of body weight, in the range of about 0.05 mg / kg to about 2 mg / kg of body weight, in the range of about 0.05 mg / kg to about 1.5 mg / kg of body weight, or in the range of about 0.05 mg / kg to about 1 mg / kg of body weight.

[0135] According to certain embodiments of the invention, an AP-based agent may be administered, for example, more than once per day (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 per day, about every other day, about every third day, about once per week, about once every two weeks, about once per three months, about once per two months, about once per three months, about once per six months, or about once per year.

[0136] Methods for Treating, Preventing, or Reducing Side Effects of Chemotherapeutic Treatment - Patent application In some embodiments, a method is provided for treating or preventing at least one side effect of a nucleoside analogue chemotherapy treatment in a subject, the method comprising administering an IAP to the subject, the subject having undergone or being treated for cancer using a nucleoside analogue chemotherapy treatment, the IAP being administered simultaneously, intermittently, or any combination thereof with respect to the nucleoside analogue chemotherapy treatment.

[0137] In various embodiments, the present invention provides a method of treating, preventing, reducing, or eliminating a side effect of a chemotherapy treatment in a cancer patient, comprising administering an IAP to the patient. In a non-limiting example, the IAP comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 41. In a further non-limiting example, the chemotherapy treatment comprises a nucleoside analog, optionally 5-FU. In another non-limiting example, the side effect of the chemotherapy treatment is diarrhea.

[0138] In various embodiments, a method of treating or preventing at least one side effect of a chemotherapeutic treatment with a nucleoside analog in a subject is provided, comprising administering an IAP to the subject, wherein the subject has undergone or is undergoing cancer treatment using a chemotherapeutic treatment. In some embodiments, the effect of administering the IAP is compared to the side effect profile of a subject undergoing a chemotherapeutic treatment but not receiving the IAP. In a non-limiting example, the IAP comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 41. In a further non-limiting example, the chemotherapeutic treatment comprises a nucleoside analog, optionally 5-FU. In another non-limiting example, the side effect of the chemotherapeutic treatment is diarrhea.

[0139] In some embodiments, the IAP is administered sequentially with the administration of the nucleoside analog, which may be before the administration of the nucleoside analog or after the administration of the nucleoside analog. In some cases, the administration of the IAP may at least partially overlap with the administration of the nucleoside analog. In some embodiments, the IAP is administered simultaneously with the administration of the nucleoside analog. For example, in some embodiments, the administration of the IAP can begin before the nucleoside analog treatment (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days before or more than 10 days before the administration of the nucleoside analog begins) and can continue throughout the entire nucleoside analog treatment. In some embodiments, the administration of the IAP (which can begin before or after the start of the administration of the nucleoside analog) can continue even after the administration of the nucleoside analog is completed. For example, administration of the IAP can continue for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 days after administration of the nucleoside analog to the subject is completed. In other embodiments, the IAP is administered intermittently with administration of the nucleoside analog.

[0140] In some cases, side effects, including gastrointestinal (GI) side effects, of chemotherapy agents, such as 5-FU, can be severe and life-threatening. GI side effects include, for example, diarrhea, loss of appetite, nausea, vomiting, stomatitis, and esophagopharyngitis, as well as various other side effects. For example, mucositis and diarrhea are common dose-limiting GI side effects. Various side effects associated with cancer treatment using nucleoside analogues can also limit the frequency and duration of chemotherapy, thereby hindering the success of cancer treatment.

[0141] Thus, the methods according to some embodiments of the present disclosure can prevent, reduce, or eliminate one or more side effects associated with chemotherapy treatment, including GI side effects. Non-limiting examples of side effects of chemotherapy treatment include diarrhea, colitis, mucositis, weight loss, pain, nausea, vomiting, constipation, anemia, malnutrition, alopecia, bone marrow suppression, nephrotoxicity, hair loss, numbness, altered taste, loss of appetite, thin or brittle hair, stomatitis, memory loss, bleeding, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment.

[0142] Methods according to embodiments of the present disclosure include administration of an effective dose of an IAP, which can reduce the likelihood of side effects occurring, as well as their duration and / or severity, and may accelerate the rate at which a patient recovers from symptoms of the side effects.

[0143] The IAP that may be orally administered may be selected from human IAP or calf / bovine IAP. In some embodiments, 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: 1-11, 17, 18, or 41. In some embodiments, an 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. In some embodiments, an IAP comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 2, or SEQ ID NO: 5, or SEQ ID NO: 41.

[0144] For example, in some embodiments, the methods according to some embodiments of the present disclosure can prevent, reduce, or eliminate the side effects of chemotherapy treatment, including diarrhea. In some embodiments, the Bristol stool scale is used to determine whether the stool of the subject administered with IAP tends to return from diarrhea (e.g., a score of 6 or 7) to normal (e.g., a score of 3 or 4). In various embodiments, the stool of the subject undergoing chemotherapy treatment scores 6 or 7 on the Bristol stool scale. In some embodiments, the subject undergoing chemotherapy treatment and administered with IAP scores 3, 4, or 5 on the Bristol stool scale.

[0145] The nucleoside analog may be any suitable nucleoside analog, including, but not limited to, 5-fluorouracil (5-FU), 5'-deoxyfluorouridine, 5-fluoro-2'-deoxyuridine triphosphate, fluorouridine, 2'-deoxyfluorouridine, fluorocytosine, trifluoro-methyl-2'-deoxyuridine, cytarabine, cyclocytidine, arabinosyl cytosine, 5-aza-2'-deoxycytidine, arabinosyl-5-azacytosine, 6-azacytidine, N-phosphonoacetyl-L-aspartic acid, pyrazofurin, 6-azauridine, azaribine, thymidine, Fazarabine, and 3-deazauridine. In some embodiments, the nucleoside analog comprises at least one of fluoropyrimidine, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, 5-fluoro-2'-deoxyuridine triphosphate, gemcitabine, troxacitabine, decitabine, cytarabine, pseudoisocytidine, azacitidine, decitabine, zebularine, ancitabine, fazarabine, 6-azacytidine, capecitabine, N4-octadecyl-cytarabine, elaidic acid-cytarabine, cladribine, acyclovir, clofarabine, nelarabine, forodesine, 8-chloroadenosine, sapacitabine, thiarabine, and derivatives thereof. In at least some embodiments, the nucleoside analog is a pyridine analog, such as 5-FU.

[0146] In some embodiments, the nucleoside analog is 5-fluorouracil (5-FU). In some embodiments, a standard 5-FU treatment regimen includes administering about 12 mg / kg once a day for 4 consecutive days, and optionally administering about 6 mg / kg on days 6, 8, 10, and 12 unless toxicity occurs. In various embodiments, administration of an IAP may allow a subject to administer the complete regimen without stopping treatment due to side effects. In some embodiments, administration of an IAP may allow a patient to administer an increased dosage (e.g., greater than about 20 mg / kg once a day, or about 18 mg / kg once a day, or about 16 mg / kg once a day, or about 14 mg / kg once a day, or about 12 mg / kg once a day) for more than 4 consecutive days (e.g., about 10, about 9, about 8, about 7, about 6, or about 5 days), greater than about 15 mg / kg once a day, or about 1 It allows for administration of 2 mg / kg once a day, or about 10 mg / kg once a day, or about 8 mg / kg once a day, or about 6 mg / kg once a day on days 6, 8, 10, and 12, and / or for a period of administration (e.g., greater than 12 days, including, for example, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24 days, about 4 weeks, or about 1 month).

[0147] In some embodiments, administration of an IAP may allow a patient to receive a dose of 5-FU of greater than about 200 mg per day, about 300 mg per day, about 400 mg per day, about 500 mg per day, about 600 mg per day, about 700 mg per day, or about 800 mg per day, or about 900 mg per day, or about 1000 mg per day.

[0148] The method of treating or preventing at least one side effect of chemotherapy treatment with nucleoside analogues in a subject can be used to treat various cancers, including colorectal cancer, breast cancer, intestinal cancer, stomach cancer, colon cancer, anal cancer, head and neck cancer, skin cancer, esophageal (throat) cancer, and pancreatic cancer. The nucleoside analogue can be administered in combination (co-administration) with one or more other nucleoside analogues or other therapeutic agents. For example, in some embodiments, the method further comprises administering one or more corticosteroids.

[0149] In some aspects, administration of IAP allows for a reduction in the dose, duration, and / or frequency of administering nucleoside analogues. In some embodiments, the reduced dose of nucleoside analogues can be a sub-therapeutic dose of nucleoside analogues. In some embodiments, use of IP allows for an increase in the therapeutic range of nucleoside analogues. The increased therapeutic range of nucleoside analogues can include one or more of: an increase in the likelihood that a subject will receive nucleoside analogue maintenance therapy; an increase in the likelihood that a subject will receive a full regimen of nucleoside analogues; an increase in the likelihood that a subject will receive more than a full regimen of nucleoside analogues; and an increase in the dose, duration, and / or frequency of nucleoside analogue treatment.

[0150] In some embodiments, administration of an effective amount of an IAP increases the ability of a cancer patient to undergo a full regimen of combination therapy with 5-FU and not have to stop treatment due to side effects, such as those discussed in more detail below. In some embodiments, administration of an effective amount of an IAP increases the ability of a patient to undergo combination therapy with 5-FU at higher doses or for longer periods of time. For example, a combination therapy regimen of 5-fluorouracil / leucovorin can be as follows: (a) 6-week cycle with infusional 5-fluorouracil / leucovorin: 180 mg / m over 30-90 minutes. 2IV infusion once daily (infused over 30–90 minutes) on days 1, 15, and 29, followed by leucovorin and 5-fluorouracil infusions; next cycle starting on day 43; or (b) 6-week cycle with bolus 5-fluorouracil / leucovorin: 125 mg / m 2 on days 1, 8, 15, and 22 (infused over 90 minutes), followed by a bolus dose of leucovorin and 5-fluorouracil. In various embodiments, administration of an effective amount of an IAP increases the dose and / or frequency of the regimen. For example, the regimen can be extended beyond 6 weeks to about 45 days, or about 50 days, or about 55 days, or about 60 days, or about 65 days, or about 70 days, or about 75 days, or about 100 days, including about 7 weeks, or about 8 weeks, or about 9 weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks.

[0151] In some embodiments, administration of an effective amount of an IAP increases the ability of a cancer patient to receive a full regimen of any one of FOLFOX, FOLFIRI, IFL, FL (Mayo), QUASAR, Machover schedule, CAF, CMF, ECF, and FEC regimens and not have to stop treatment due to side effects. In some embodiments, administration of an effective amount of an IAP increases the ability of a cancer patient to receive a larger dose or longer duration of therapy with FOLFOX, FOLFIRI, IFL, FL (Mayo), QUASAR, Machover schedule, CAF, CMF, ECF, and FEC regimens.

[0152] In some embodiments, the methods contemplate preventing or reducing a reduction in gut microbiome diversity that may be a side effect or consequence of chemotherapy treatment, hi some embodiments, the methods relate to repairing and / or repopulating the gut microbiome in a subject following chemotherapy treatment.

[0153] In various embodiments, the method prevents, reduces, or eliminates the side effects of chemotherapy treatment, including acute side effects, long-term side effects, or cumulative side effects. In various embodiments, the method reduces or eliminates the local or systemic side effects of chemotherapy treatment. In various embodiments, the side effects of chemotherapy treatment are one or more of diarrhea, colitis, mucositis, weight loss, pain, nausea, vomiting, constipation, anemia, malnutrition, alopecia, bone marrow suppression, nephrotoxicity, hair loss, numbness, change in taste, loss of appetite, thin or brittle hair, stomatitis, memory loss, bleeding, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment. Side effects may also include fatigue, epithelial surface damage infertility, fibrosis, hair loss, dryness (e.g., but not limited to, dry mouth (xerostomia) and dry eyes (xerophthalmia), and dry axillary and vaginal mucosa), lymphedema, cardiac disease, cognitive decline, and vitamin B12 malabsorption.

[0154] In some embodiments, the IAP is administered in combination with an additional agent, which can be any of the agents described herein or another agent, including, but not limited to, a corticosteroid, an antioxidant (e.g., amifostine and vitamin E), a cytokine (e.g., stem cell factor), and the like.

[0155] In various embodiments, administration of an AP-based agent stimulates and protects stem cells. For example, the present invention and compositions can stimulate and protect hematopoietic stem cells, including various hematopoietic progenitor cells. In another example, the present methods and compositions can stimulate and protect gastrointestinal stem cells, such as epithelial cells (e.g., goblet cells) and intestinal crypt stem cells. In some embodiments, the present methods and compositions protect small intestinal cells, such as goblet cells, from hypoplasia and loss (e.g., apoptosis or necrosis). In various embodiments, the present methods and compositions significantly enhance the recovery of the GI system from chemotherapy drug treatment.

[0156] In various embodiments, the subject is a cancer patient. Cancers include, but are not limited to, colon and rectal cancer; basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including GI cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, Lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; Hodgkin's and non-Hodgkin's lymphoma, and B-cell lymphoma Lymphomas, including follicular lymphoma (including 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 immunoblastic NHL; high grade small noncleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's hypergammaglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel growth associated with nematoses, edema (such as associated with brain tumors) or Meigs syndrome.

[0157] Additional Therapeutic Agents and Combination Therapies The administration of the compositions and formulations comprising an AP-based agent can be combined with an additional agent. The co-administration of the additional agent with the composition / formulation can be simultaneous, sequential, or a combination thereof. Additionally, the composition / formulation can include an additional agent (e.g., via co-formulation). For example, the additional agent and the AP-based agent can be combined in a single formulation. Alternatively, the additional agent and the AP-based agent can be formulated separately.

[0158] In one embodiment, the additional agent and the AP-based agent are administered to the subject simultaneously. The term "simultaneously" as used herein means that the additional agent and the AP-based agent are administered at a time interval of about 60 minutes or less, for example, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, about 5 minutes or less, or about 1 minute or less. The administration of the additional agent and the AP-based agent can be simultaneous administration in a single formulation (e.g., a formulation that includes the additional agent and alkaline phosphatase) or simultaneous administration in separate formulations (e.g., a first formulation that includes the additional agent and a second formulation that includes the AP-based agent).

[0159] In further embodiments, the additional agent and the AP-based agent are administered to a subject simultaneously, but release of the additional agent and alkaline phosphatase from the corresponding dosage form (or single unit dosage form if co-formulated) may occur sequentially.

[0160] Concurrent administration does not require that the additional agent and the AP-based agent be administered at the same time, provided that the timing of administration is such that the pharmacokinetic activities of the additional agent and the AP-based agent overlap over time. For example, the additional agent and the AP-based agent can be administered sequentially. The term "sequential" as used herein means that the additional agent and the AP-based agent are administered at a time interval of more than about 60 minutes. For example, the time between the sequential administration of the additional agent and the AP-based agent can be more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, or more than about 1 week. The administration time depends on the metabolic rate, excretion, and / or pharmacodynamic activity of the additional agent and the AP-based agent administered. Either the additional agent or the AP-based agent can be administered first.

[0161] Concomitant administration also does not require that the additional agent and the AP-based agent be administered to the subject by the same route of administration. Rather, each therapeutic agent can be administered by any suitable route, for example, parenterally or non-parenterally.

[0162] In various embodiments, the additional agents include one or more of blood products, colony stimulating factors, cytokines and / or growth factors, antibiotics, diluting and / or blocking agents, mobilizing or chelating agents, stem cell transplants, and antioxidants or free radicals, and in embodiments where chemotherapy is used in combination with radiation therapy, the additional agents include various radiation protectants.

[0163] In some embodiments, the blood product is a hematopoietic growth factor, such as filgrastim (e.g., NEUPOGEN), granulocyte colony-stimulating factor (G-CSF), which may optionally be pegylated (e.g., NEULASTA), sargramostim (LEUKINE), and one or more of granulocyte-macrophage colony-stimulating factor (GM-CSF) and KSF.

[0164] In some embodiments, the additional agent is one or more of a cytokine and / or growth factor that may confer cytoprotection by replenishing and / or protecting the stem cell population. In some cases, stem cell factor (SCF, c-kit ligand), Flt-3 ligand, and / or interleukin 1 fragment IL-1b-rd may be used. Some factors are not cytokines in nature, but stimulate the proliferation of immune cells and can be used in combination with AP-based agents. For example, 5-AED (5-androstenediol) is a steroid that stimulates the expression of cytokines to increase resistance to bacterial and viral infections. Synthetic compounds, such as ammonium trichloro(dioxoethylene-O,O'-) tellurate (AS-101), can also be used to induce the secretion of a number of cytokines and in combination with AP-based agents. Growth factors and cytokines can also be used to provide protection against gastrointestinal syndrome. Keratinocyte growth factor (KGF) promotes proliferation and differentiation in the intestinal mucosa and increases cell survival after irradiation in the intestinal crypts. The hematopoietic cytokine SCF may also increase intestinal stem cell survival and associated short-term organism survival.

[0165] In certain embodiments, the AP-based agent is administered in combination with a cytokine regimen (e.g., FILGRASTIM (G-CSF) at 2.5-5 μg / kg / day once daily, subcutaneously (100-200 μg / m 2 / day), and SARGRAMOSTIM (GM-CSF), 5-10 μg / kg / day, once daily, subcutaneously (200-400 μg / m 2 / day), and / or PEGFILGRASTIM (pegG-CSF), 6 mg once, subcutaneously).

[0166] In some embodiments, the additional agent is an interleukin, such as IL-12 (eg, HEMAMAX (NEUMEDICINES, INC)).

[0167] In some embodiments, the antibiotic is one or more of an antibacterial (anti-gram positive and anti-gram negative), and / or an antifungal, and / or an antiviral. As non-limiting examples, in some embodiments, the antibiotic can be a quinolone, such as ciprofloxacin, levofloxacin, a third or fourth generation cephalosporin with pseudomonal range, such as cefepime, ceftazidime, or an aminoglycoside, such as gentamicin, amikacin, penicillin or amoxicillin, acyclovir, vancomycin. In various embodiments, the antibiotic targets Pseudomonas aeruginosa.

[0168] In some embodiments, the additional agent is a stem cell transplant (e.g., bone marrow transplant, PBSCT, MSCT). In some embodiments, the stem cell transplant is CTL-008 (Cellerant), Remestemcel-L (Osiris).

[0169] In some embodiments, the additional agent is an antioxidant or free radical. Antioxidants and free radical scavengers that can be used in the practice of the present invention include, but are not limited to, thiols such as cysteine, cysteamine, glutathione, and bilirubin; amifostine (WR-2721); vitamin A; vitamin C; vitamin E; and flavonoids such as Indian holy basil (Ocimum sanctum), orientin, and vicenin.

[0170] In some embodiments, the additional agent may be a steroid (e.g., 5-androstenediol), an antioxidant (e.g., amifostine, vitamin E, gamma tocotrienol (a vitamin E moiety), and genistein (a soy by-product)), a cytokine (e.g., stem cell factor), a growth factor (e.g., keratinocyte growth factor), ammonium trichloro(dioxoethylene-O,O') tellurate, an antiemetic, an antidiarrheal, an antiemetic (e.g., an oral prophylactic antiemetic, such as granisetron (KYTRIL), ondansetron (ZOFRAN), and a 5-HT3 blocker with or without dexamethasone), analgesic, anxiolytic, sedative, cytokine therapy, and an antibiotic.

[0171] Further examples of combination agents include alkylating agents such as thiotepa and CYTOXAN cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; statins; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and biceresin); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, coumadin, nitrogen mustards, such as cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Enediyne antibiotics, such as the calicheamicins, especially calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicins; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromogens. group), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norubicin, leucine, adremiamycin, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, queramycin antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; def of amine amine);demecolcine;diaziquone;elformithine;elliptinium acetate;epothilones;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidainine;maytansinoids such as maytansine and ansamitocin;mitoguazone;mitoxantrone;mopidanmol;nitraerine;pentostatin;phenamet;pirarubicin;losoxantrone;podophyllinic acid;2-ethylhydrazide;procarbazine;PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE Cremophor-free albumin-modified nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE docetaxel (Rhone-Poulenc Rorer,Antony,France);Chlorambucil;GEMZAR gemcitabine;6-thioguanine;Mercaptopurine;Methotrexate;Platinum analogues such as cisplatin, oxaliplatin, and carboplatin;Vinblastine;Platinum;Etoposide (VP-16);Ifosfamide;Mitoxantrone;Vincristine;NAVELBINE.Vinorelbine;Novantrone;Teniposide;Edatrexate;Daunomycin;Aminopterin;Xeloda;Ibandronate;Irinotecan (Camptosar, CPT-11) (5- including irinotecan with FU and leucovorin treatment regimens); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin including oxaliplatin treatment regimens (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation, as well as pharmacologic acceptable salts, acids, or derivatives of any of the above. In addition, the method of treatment can further include the use of radiation. In addition, the method of treatment can further include the use of photodynamic therapy.;

[0172] definition As used herein, "a," "an," or "the" can mean one or more than one.

[0173] Furthermore, the term "about," when used in connection with a reference numerical indication, means the reference numerical indication plus or minus up to 10% of the reference numerical indication. For example, the term "about 50%" covers a range of 45% to 55%.

[0174] An "effective amount," when used in relation to medical applications, is an amount effective to provide a measurable treatment, prevention, or reduction in the rate of pathogenesis of the disorder of interest.

[0175] As used herein, something is "decreased" if an activity and / or effect readout is reduced by a significant amount in the presence of an agent or stimulus compared to the absence of such modulation, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to at least about 100%. As will be appreciated by one of skill in the art, in some embodiments, activity is decreased and some downstream readouts are decreased while others may be increased.

[0176] Conversely, an activity is "increased" if a readout of the activity and / or effect is increased by a significant amount in the presence of an agent or stimulus compared to the absence of such agent or stimulus, e.g., by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to at least about 100%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold.

[0177] When referred to herein, all compositional percentages are based on the weight of the entire composition unless otherwise specified. As used herein, the word "comprises" and variations thereof are intended to be open-ended, with the recitation of the items in the list not excluding other similar items that may also be useful in the compositions and methods of the present technology. Similarly, the terms "can" and "may" and variations thereof are intended to be open-ended, with the recitation of an embodiment that may or may include certain elements or features not excluding other embodiments of the present technology that do not contain these elements or features.

[0178] Although the open-ended term "comprising," which is synonymous with terms such as including, containing, or having, is used herein to describe and claim the invention, the invention or embodiments thereof may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

[0179] As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that provide certain benefits, under certain conditions. However, other embodiments may 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.

[0180] In some embodiments, the terms "patient" and "subject" are used interchangeably. In some embodiments, the subject and / or animal is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, goat, or a non-human primate, such as a monkey, chimpanzee, or baboon. In other embodiments, the subject and / or animal is a non-mammal, such as a zebrafish.

[0181] In various embodiments, the methods of the invention are useful for treating a human subject. In some embodiments, the human is a child. In some embodiments, the human is an adult. In other embodiments, the human is an elderly person. In other embodiments, the human may also be referred to as a patient. In some embodiments, the human is a female. In some embodiments, the human is a male.

[0182] In certain embodiments, the human has an age ranging from about 1 to about 18 months, about 18 to about 36 months, about 1 to about 5 years, about 5 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, about 20 to about 25 years, about 25 to about 30 years, about 30 to about 35 years, about 35 to about 40 years, about 40 to about 45 years, about 45 to about 50 years, about 50 to about 55 years, about 55 to about 60 years, about 60 to about 65 years, about 65 to about 70 years, about 70 to about 75 years, about 75 to about 80 years, about 80 to about 85 years, about 85 to about 90 years, about 90 to about 95 years, or about 95 to about 100 years.

[0183] The amount of the compositions described herein necessary to achieve a therapeutic effect can be empirically determined according to a procedure convenient for a particular purpose. In general, for administration of a therapeutic agent (e.g., a microbiome modulating agent and / or an additional therapeutic agent described herein) for therapeutic purposes, the therapeutic agent is given in a pharmacologically effective dose. A "pharmacologically effective amount", "pharmacologically effective dose", "therapeutically effective amount", or "effective amount" refers to an amount sufficient to produce a desired physiological effect or to achieve a desired result, in particular an amount capable of treating a disorder or disease. An effective amount, as used herein, includes, for example, an amount sufficient to delay the onset of a symptom of a disorder or disease, to alter the course of a symptom of a disorder or disease (e.g., to slow the progression of a symptom of a disease), to reduce or eliminate one or more symptoms or signs of a disorder or disease, and to reverse a symptom of a disorder or disease. A therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether an improvement is realized.

[0184] Effective doses, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates, or experimental animals, for example, to determine the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). Dosages can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. In some embodiments, compositions and methods that exhibit large therapeutic indices are preferred. Therapeutically effective doses can be estimated initially, for example, in cell culture assays or measurements, or in vitro assays including methane production in stool samples. Doses can also be formulated in cell culture or animal models to achieve a circulating plasma concentration range that includes the IC50 determined in a suitable animal model. Levels of the compositions described herein in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by a suitable bioassay. Dosage will be determined by the physician and can be adjusted, if necessary, to conform to the observed effects of the treatment.

[0185] In certain embodiments, the effect is 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% quantitative change. In some embodiments, the effect is at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or even at least about 90% quantitative change. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is achieved.

[0186] As used herein, "method of treatment" applies equally to the use of a composition to treat a disease or disorder described herein and / or the composition for use and / or in the manufacture of a medicament to treat a disease or disorder described herein.

[0187] Working Example Example 1: IAP reduces the side effects of 5-FU Experiments were conducted to evaluate the effect of IAP treatment on the side effects of diarrhea and weight change associated with chemotherapy agents containing nucleoside analogues for the treatment of cancer.

[0188] The study performed consisted of evaluating the effect of administration of IAP ("SYN-020" is synonymous with IAP and SYN BIAPII) with chemotherapy treatment with 5-fluorouracil (5-FU) in a murine model. In summary, Figures 1A-1B and 2A-2C illustrate that administration of IAP reduces the side effects of 5-FU based on the evaluation of mouse weight change and stool consistency. Stool consistency was scored as follows: score 0 = normal (no diarrhea), score 2 = soft, formed stool, and score 4 = diarrhea.

[0189] The first set of experiments consisted of three groups of BALB / c mice inoculated with CT26 murine colorectal cancer cells in the right flank: (1) an untreated control group of 18 mice ("Untreated"), (2) a group of 18 male mice that received an intraperitoneal (IP) QD (once daily) dose of 30 mg / kg 5-FU for five consecutive days (days 0-4) and vehicle administered PO (orally) BID (twice daily) throughout the study (days -3 to +26) ("Vehicle + 5-FU"), and (3) a group of 18 male mice that received an IP QD dose of 30 mg / kg 5-FU for five consecutive days (days 0-4) and IAP (SYN-020) at a dose of 0.5 mg / kg administered PO BID throughout the study (days -3 to +26) ("SYN BIAPII + 5-FU").

[0190] FIG. 1A shows the mean percent weight change ± SEM for untreated (upper curve), vehicle + 5-FU (middle curve), and SYN BIAPII + 5-FU (lower curve) mice versus days of study. FIG. 1B shows stool consistency assessment by mean stool consistency score ± SEM versus days for vehicle + 5-FU and SYN BIAPPII + 5-FU mice compared to untreated mice data shown at baseline on the x-axis. As shown, administration of 5-FU caused loose stools / diarrhea, as well as associated weight loss, in all treated animals. These symptoms peaked on day 6 and resolved by day 9 in both vehicle + 5-FU and SYN BIAPII + 5-FU mouse groups. Administration of IAP in the SYN BIAPII + 5-FU group shows significantly faster recovery from loose stool / diarrhea symptoms and associated weight loss (p<0.03).

[0191] The second set of experiments consisted of four groups of BALB / c mice inoculated with CT26 murine colorectal cancer cells in the right flank. (2) a group of 30 male mice that received a daily therapeutic dose of SYN BIAPII only at 100 U / dose, 0.5 mg / kg ("SYN BIAPII"); (3) a group of 30 male mice that received an IP QD dose of 30 mg / kg of 5-FU for five consecutive days (days 0-4) plus vehicle treatment ("Vehicle+5-FU"); and (4) a group of 30 male mice that received an IP QD dose of 30 mg / kg of 5-FU for five consecutive days (days 0-4) plus IAP (SYN-020) at a dose of 100 U / dose, 0.5 mg / kg, administered PO BID throughout the study (days -3 to +26) ("SYN BIAPII+5-FU").

[0192] Figure 2A depicts mean daily body weights at 50% group survival, shown as percent change over time compared to day 0 body weight + SEM. Animals treated with vehicle + 5-FU from days 0 to 4 showed chemotherapy-induced body weight loss, peaking at approximately (-20%) on day 7, after which animals regained lost body weight by day 20 of drug. The mean percentage body weight change for animals treated with SYN BIAPII + 5-FU was not significantly different from animals treated with vehicle + 5-FU. Differences in cumulative body weight change (days (-3) to 16) between treatment groups at 50% general group survival were assessed by calculating the area under the curve (AUC) for each animal using a trapezoidal transformation.

[0193] Figure 2B shows the results of daily stool consistency scores from days 0 to 12, with the mean scores + SEM shown. Arrows indicate the days of 5-FU administration. Animals treated with vehicle or SYN BIAPII alone generally showed normal stool consistency throughout the analysis. In animals treated with vehicle + 5-FU, or SYN BIAPII + 5-FU, the occurrence of loose stools or diarrhea was first observed on day 5. In animals treated with vehicle + 5-FU, the occurrence of loose stools generally resolved by day 12, and in animals treated with SYN BIAPII + 5-FU, the occurrence of loose stools generally resolved by day 11. Compared to treatment with vehicle + 5-FU, an improvement in overall stool scores was observed over days 5 to 11 in animals treated with SYN BIAPII + 5-FU. Statistically significant group differences in daily stool scores were assessed daily by Mann-Whitney rank sum analysis. Compared to treatment with vehicle+5-FU, animals treated with SYN BIAPPII+5-FU showed a statistically significant improvement in overall stool scores at day 5 (p=0.006).

[0194] Table 1 below compares the duration of 5-FU induced diarrhea by calculating the percentage of days animals had diarrhea (score=4) relative to the total. Groups were compared by chi-square test. Compared to untreated animals, a statistically significant cumulative increase in the number of days animals had diarrhea was observed in both vehicle+5-FU treated animals (p≦0.0001) and SYN BIAPPII+5-FU treated animals (p≦0.0001). Compared to treatment with vehicle+5-FU, animals treated with SYN BIAPPII+5-FU showed a statistically significant improvement in overall diarrhea duration (p=0.0006).

[0195] [Table 12]

[0196] Finally, FIG. 2C depicts a graphical representation of the percentage of days animals had diarrhea by treatment group.

[0197] Thus, this study demonstrates that administration of an IAP ameliorates the side effects associated with 5-FU, such as diarrhea and mucositis, and therefore, IAPs can be utilized as therapeutic agents used to treat, prevent, and reduce cancer therapy-related side effects, as well as to enhance the overall response of chemotherapeutic agents.

[0198] Equivalent While the invention has been described in relation to particular embodiments, it will be understood that further modifications are possible, and that this application is intended to cover any variation, use, or adaptation of the invention which generally follows from the principles of the invention and includes departures from the present disclosure as may be applied to the essential features described hereinabove and as are known or customary practice within the art to which the invention pertains, and which comply with the scope of the appended claims.

[0199] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed within the scope of the following claims.

[0200] Incorporated by reference All patents and publications cited herein are hereby incorporated by reference in their entirety.

[0201] 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 invention is not entitled to antedate such publication by virtue of prior invention.

[0202] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may be equally applicable to all sections. (Additional Note) The present disclosure includes the following aspects. Item 1: A method for treating or preventing at least one side effect of a chemotherapeutic treatment with a nucleoside analog in a subject, the method comprising administering an intestinal alkaline phosphatase (IAP) to the subject, wherein the subject has undergone or is undergoing cancer treatment using the chemotherapeutic treatment. Item 2: The method of item 1, wherein the IAP is administered sequentially with the chemotherapeutic treatment. Item 3: The method of item 2, wherein the IAP is administered prior to the chemotherapeutic treatment. Item 4: The method of item 2, wherein the IAP is administered after the chemotherapy treatment. Item 5: The method of item 1, wherein the IAP is administered simultaneously with the chemotherapeutic treatment. Item 6: The method of item 1, wherein the IAP is administered intermittently with the chemotherapy treatment. Item 7: The method according to any one of items 1 to 6, wherein the side effects of the chemotherapy drug treatment include diarrhea, colitis, mucositis, weight loss, pain, nausea, vomiting, constipation, anemia, malnutrition, alopecia, bone marrow suppression, nephrotoxicity, hair loss, numbness, altered taste, loss of appetite, thinning or brittle hair, stomatitis, memory loss, bleeding, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment. Item 8: The method of any one of items 1 to 7, wherein the side effects of the chemotherapy treatment include gastrointestinal (GI) side effects. Item 9: The method of items 1 or 8, wherein the side effect comprises mucositis. Item 10: The method of any one of items 1 to 9, wherein the side effect comprises diarrhea. Item 11: The method of any one of items 1 to 10, wherein the IAP is selected from human IAP or calf / bovine IAP. Item 12: The method of any one of items 1 to 11, 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: 1-11, 17, 18, or 41. Item 13: The method of any one of items 1 to 11, 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 NO:2, SEQ ID NO:5, or SEQ ID NO:41. Item 14: The method of any one of items 1 to 11, wherein the IAP comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 41. Item 15: The method according to any one of items 1 to 14, wherein the IAP is administered orally. Item 16: The method of any one of items 1 to 15, wherein the IAP is formulated for gastrointestinal (GI) release. Item 17: The method according to any one of items 1 to 16, wherein the nucleoside analog is a purine or a pyrimidine analog or derivative thereof. Item 18: The method of item 1, wherein the nucleoside analog is a pyrimidine analog. Item 19: The method of item 1, wherein the pyrimidine analog comprises at least one of 5-fluorouracil (5-FU), 5'-deoxyfluorouridine, 5-fluoro-2'-deoxyuridine triphosphate, fluorouridine, 2'-deoxyfluorouridine, fluorocytosine, trifluoro-methyl-2'-deoxyuridine, cytarabine, cyclocytidine, arabinosyl cytosine, 5-aza-2'-deoxycytidine, arabinosyl-5-azacytosine, 6-azacytidine, N-phosphonoacetyl-L-aspartic acid, pyrazofurin, 6-azauridine, azaribine, thymidine, fazarabine, 3-deazauridine, and derivatives thereof. Item 20: The method of item 19, wherein the pyrimidine analog is 5-fluorouracil. Item 21: The method according to any one of items 1 to 16, wherein the nucleoside analog comprises at least one of fluoropyrimidine, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, 5-fluoro-2'-deoxyuridine triphosphate, gemcitabine, troxacitabine, decitabine, cytarabine, pseudoisocytidine, azacitidine, decitabine, zebularine, ancitabine, fazarabine, 6-azacytidine, capecitabine, N4-octadecyl-cytarabine, elaidic acid-cytarabine, cladribine, acyclovir, clofarabine, nelarabine, forodesine, 8-chloroadenosine, sapacitabine, thiarabine, and derivatives thereof. Item 22: The method according to any one of Items 1 to 21, wherein the subject has cancer. Item 23: The cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, cancer of the brain and central nervous system, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, cancer of the colon and rectum, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including GI cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g. small cell lung cancer, non-small cell Lung cancer, lung adenocarcinoma and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; Hodgkin's and non-Hodgkin's lymphoma, and B-cell lymphoma 23. The method of claim 22, comprising treating lymphomas including lymphomas (including 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 immunoblastic NHL; high grade small noncleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's hypergammaglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nematoses, edema (such as associated with brain tumors) or Meigs' syndrome. Item 24: The method according to any one of items 1 to 23, wherein administration of the IAP allows for a reduction in the dose, duration and / or frequency of administration of the nucleoside analogue. Item 25: The method of item 24, wherein the reduced dose of the nucleoside analog is a sub-therapeutic dose of the nucleoside analog. Item 26: The method of any one of items 1 to 25, wherein the method increases the therapeutic range of the nucleoside analogue. Item 27: The method of item 26, wherein the increased therapeutic range of the nucleoside analog comprises one or more of: an increased likelihood that the subject will receive nucleoside analog maintenance therapy, an increased likelihood that the subject will receive a full regimen of the nucleoside analog, an increased likelihood that the subject will receive more than a full regimen of the nucleoside analog, and an increased dose, duration and / or frequency of nucleoside analog treatment. Item 28: The method of any one of items 1 to 27, wherein the IAP does not interfere with the cancer treatment in the subject. Item 29: 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: 41; the chemotherapy treatment is 5-FU; the side effects of the chemotherapy treatment include gastrointestinal (GI) side effects; The method according to item 1. Item 30: The method of item 29, wherein the gastrointestinal (GI) side effect is diarrhea. Item 31: The method of item 29, wherein the gastrointestinal (GI) side effect is mucositis. Item 32: 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: 41; the chemotherapeutic treatment is 5-FU; the side effects of the chemotherapy treatment include gastrointestinal (GI) side effects; The method according to any one of items 1 to 31. Item 33: The method of item 32, wherein the gastrointestinal (GI) side effect is diarrhea. Item 34: The method of item 32, wherein the gastrointestinal (GI) side effect is mucositis.

Claims

1. 1. A pharmaceutical composition for treating or preventing at least one side effect of a chemotherapeutic treatment with a nucleoside analog in a subject, comprising an intestinal alkaline phosphatase (IAP) inhibitor, the subject has undergone or is undergoing cancer treatment using the chemotherapeutic treatment; The IAP comprises an amino acid sequence having at least 90% identity to SEQ ID NO:41; the nucleoside analog comprises 5-fluorouracil; At least one side effect of the chemotherapeutic treatment includes diarrhea and / or mucositis; Pharmaceutical compositions.

2. (i) the IAP is administered sequentially with the chemotherapeutic treatment; or (ii) the IAP is administered simultaneously with the chemotherapeutic treatment; or (iii) the IAP is administered intermittently with the chemotherapy treatment; The pharmaceutical composition of claim 1.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the at least one side effect of the chemotherapy treatment further comprises colitis, weight loss, pain, nausea, vomiting, constipation, anemia, malnutrition, alopecia, bone marrow suppression, nephrotoxicity, hair loss, numbness, altered taste, loss of appetite, thinning or brittle hair, stomatitis, memory loss, bleeding, cardiotoxicity, hepatotoxicity, ototoxicity, or post-chemotherapy cognitive impairment.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the IAP is administered orally.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject has cancer.

6. The cancer is basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; cancer of the brain and central nervous system; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; cancer of the colon and rectum; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; Eye cancer; head and neck cancer; gastric cancer; GI cancer; glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasm; renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer; Small cell lung cancer; Non-small cell lung cancer; Lung adenocarcinoma; Lung squamous cell carcinoma; Melanoma; Myeloma; Neuroblastoma; Oral cancer; Ovarian cancer; Pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; Cancer of the respiratory system; Cancer of the salivary glands; Sarcoma; Skin cancer; Squamous cell carcinoma; Cancer of the stomach; Testicular cancer; Thyroid cancer; Cancer of the uterus or endometrium; Cancer of the urinary system; Vulvar cancer; Lymphoma; Hodgkin's lymphoma; Non-Hodgkin's lymphoma; B-cell lymphoma; 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 immunoblastic NH The pharmaceutical composition of claim 5, comprising: L; high-grade small non-cleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's hypergammaglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; other carcinomas and sarcomas; post-transplant lymphoproliferative disorder (PTLD); or Meigs syndrome.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein said IAP allows for a reduction in the dose, duration and / or frequency of administration of said nucleoside analogue.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the pharmaceutical composition enhances the therapeutic range of the nucleoside analogue.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein said IAP does not interfere with said cancer treatment in said subject.

10. The IAP comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 41; The pharmaceutical composition of claim 1.

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