β-lactamase compositions for the treatment of graft-versus-host disease

Abstract

β-lactamase compositions for the treatment of graft-versus-host disease are provided. [Solution] The present invention relates, in part, to methods and compositions for reducing the incidence and / or severity of complications associated with the intravenous use of beta-lactam antibiotics in allo-HCT recipients, such as aGVHD and VRE colonization and / or VRE bloodstream infections, using beta-lactamase agents.

Description

[Technical Field]

[0001] The present invention relates, in part, to methods and compositions for reducing the incidence and / or severity of graft-versus-host disease (GVHD). In particular, the present invention provides, in part, methods and compositions for reducing the incidence and / or severity of GVHD using one or more β-lactamase agents.

[0002] Government Interests This invention was made with government support under Grant No. CA008748 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 843,849, filed May 6, 2019, the entire contents of which are incorporated herein by reference.

[0004] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated by reference in their entirety. A computer-readable copy of the Sequence Listing (Filename: "SYN-043PC_ST25.txt", Created: May 4, 2020, File Size: 12,140 bytes). [Background technology]

[0005] Allogeneic hematopoietic stem cell transplantation (allo-HCT) can be a curative treatment for leukemia and lymphoma and is used to treat 18,000 patients annually. However, graft-versus-host disease (GVHD) is a major complication that occurs in 20–50% of allo-HCT recipients. Approximately half of GVHD cases are steroid-responsive. Steroid-resistant GVHD, and particularly gastrointestinal (GI) GVHD, is a serious problem, accounting for approximately 20% of posttransplant mortality. Indeed, mouse studies have further demonstrated that antibiotic treatment of mice with GVHD results in the loss of the protective mucus lining of the colon, leading to intestinal barrier dysfunction.

[0006] In particular, severe dysbiosis of the gut microbiota is observed in allo-HCT patients, likely due to the broad-spectrum antibiotics frequently required for empirical treatment of fever, especially febrile neutropenia. As a result, it is thought that the gut microbiota may actually modulate the risk of infection and GVHD after allo-HCT.

[0007] Currently, there are few effective treatments aimed at reducing GI GVHD complications, and therefore new strategies to prevent or reduce GVHD are needed. Summary of the Invention [Means for solving the problem]

[0008] The present invention provides methods and compositions for reducing the incidence and / or severity of complications associated with intravenous (IV) administration of β-lactam antibiotics to allo-HCT recipients, such as, inter alia, acute GVHD (aGVHD) and / or vancomycin-resistant enterococcus (VRE) colonization and / or infection. In one aspect, the present invention provides a method for reducing the incidence and / or severity of complications associated with intravenous (IV) administration of β-lactam antibiotics to allo-HCT recipients by administering a β-lactamase agent to a subject in need thereof. In another aspect, the present invention contemplates a method for reducing the incidence and / or severity of aGVHD by administering a β-lactamase agent described herein to a subject in need thereof. In another aspect, the present invention contemplates a method for preventing VRE colonization and / or VRE bloodstream infection by administering a β-lactamase agent described herein to a subject in need thereof. In various embodiments, the subject is receiving or has been receiving intravenously (IV) administered β-lactam antibiotics.

[0009] In some embodiments, reducing the incidence and / or severity of complications associated with allo-HCT, such as aGVHD and / or VRE colonization and / or VRE bloodstream infections, includes preventing changes to the microbiome. For example, the methods of the invention can result in the prevention and / or attenuation of microbiome monodominance due to loss of microbial diversity and overgrowth by any given bacterial strain.

[0010] In various embodiments, the subject at risk for developing aGVHD is a transplant recipient. In other embodiments, the subject is a recipient of allo-HCT. In some embodiments, the subject is a recipient of one or more of bone marrow cells, peripheral blood cells, and umbilical cord blood cells.

[0011] In some embodiments, the compositions of the invention are administered prior to transplantation, hi some embodiments, the compositions of the invention are administered subsequent to transplantation.

[0012] In various embodiments, the formulations and methods described herein function exclusively in the gastrointestinal (GI) tract, thereby avoiding or reducing systemic exposure to the β-lactamase agent and providing specific delivery while minimizing its potential systemic effects. In various embodiments, the β-lactamase agent is released to one or more regions of the intestinal tract. In one embodiment, the β-lactamase agent is released into the small intestine. In another embodiment, the β-lactamase agent is released into the large intestine.

[0013] In various embodiments, the microbiome protectant (e.g., a β-lactamase agent) may be administered to a subject in combination with additional therapeutic or prophylactic agents, including, but not limited to, immunosuppressants (e.g., cyclosporine, tacrolimus, methotrexate), antibodies, and other immunomodulators that prevent aGVHD. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the reduction in piperacillin (µg per g of feces) in mice receiving 500 mg / kg piperacillin / tazobactam subcutaneously twice daily for two days and a liquid formulation of P3A (e.g., β-lactamase in phosphate-buffered saline) by oral gavage twice daily for two days. The leftmost bar corresponds to data from a cohort of control mice, the middle bar corresponds to data from a first cohort of mice receiving piperacillin / tazobactam but not P3A (e.g., β-lactamase in phosphate-buffered saline), and the rightmost bar corresponds to data from a second cohort of mice receiving both piperacillin / tazobactam and P3A (e.g., β-lactamase in phosphate-buffered saline). [Figure 2]FIG. 1 shows that administration of piperacillin / tazobactam alone reduced the abundance of Firmicutes in mouse feces, and administration of piperacillin / tazobactam with P3A restored and protected the fold abundance of Firmicutes. The leftmost bar in the bar graph corresponds to data from a cohort of control mice that received neither piperacillin / tazobactam nor β-lactamase, the second bar from the left corresponds to data from a cohort of mice that received P3A only, the second bar from the right corresponds to data from a first cohort of mice that received piperacillin / tazobactam but not a liquid formulation of P3A (e.g., β-lactamase in phosphate-buffered saline solution), and the rightmost bar in the bar graph corresponds to data from a second cohort of mice that received both piperacillin / tazobactam and P3A (e.g., β-lactamase in phosphate-buffered saline solution). [Figure 3] This figure shows that piperacillin / tazobactam reduced the total bacterial copy number present in mouse feces, and this reduction was attenuated when piperacillin / tazobactam was administered with P3A. The leftmost bar in the bar graph corresponds to data from a cohort of control mice that received neither piperacillin / tazobactam nor β-lactamase; the second bar from the left corresponds to data from a cohort of mice that received P3A only; the second bar from the right corresponds to data from a first cohort of mice that received piperacillin / tazobactam but not a liquid formulation of P3A (e.g., β-lactamase in phosphate-buffered saline); and the rightmost bar corresponds to data from a second cohort of mice that received both piperacillin / tazobactam and P3A (e.g., β-lactamase in phosphate-buffered saline). [Figure 4]

[0023] Figure 1 shows the results of a survival study with an endpoint of 30 days after bone marrow transplantation (BMT). The data demonstrate that antibiotic-exacerbated GVHD associated with bone marrow transplantation was reduced by P3A administration in terms of reduced mortality. BM refers to the cohort that received bone marrow cells only. BM+T refers to the cohort that received bone marrow cells and splenic T lymphocytes. [Figure 5] Figure 16 shows the results of 16S sequence analysis. Fecal samples from mice administered both piperacillin / tazobactam (Zosyn) and SYN-004 (P3A) showed reduced monodominance of Enterococcus compared to fecal samples from mice administered piperacillin / tazobactam alone. The saline group showed the largest bar for Clostridium, the Zosyn group showed the largest bar for Enterococcus, and the Zosyn + SYN-004 group showed the largest bar for Clostridium. [Figure 6] This figure shows the results of 16S sequencing analysis. Fecal samples from mice administered both piperacillin / tazobactam (Zosyn; abbreviated ZO) and SYN-004 (P3A; abbreviated Bact) showed reduced monodominance of Enterococcus compared to fecal samples from mice administered ZO alone. Open bars indicate that a particular sample was not amplified. In the bone marrow (BM) group, the saline cohort showed the largest bar for Clostridium, the ZO cohort showed the largest bar for Enterococcus, and the ZO + Bact cohort showed the largest bar for Enterococcus in the first and second bars from the left and the largest bar for Clostridium in the third and fourth bars from the left. In the bone marrow (BM) + T cell group, the saline cohort showed the largest bar for Clostridium, the ZO cohort showed the largest bar for Enterococcus, and the ZO + Bact cohort showed the largest bar for Clostridium. [Figure 7] FIG. 1 shows restoration and protection of Firmicutes fold abundance in the presence of both imipenem + cilastatin and 10 mg / kg and 50 mg / kg doses of P2A compared to the presence of imipenem + cilastatin alone. [Figure 8]This figure shows the results of 16S sequencing. Fecal samples from mice receiving both imipenem + cilastatin and SYN-006 (P2A; 10 mg / kg and 50 mg / kg doses) showed reduced monodominance of Enterococcus compared to fecal samples from mice receiving only imipenem + cilastatin. In the imipenem group, the pre-antibiotic ("pre-abx") cohort showed the largest bar for Lactobacillus, while the post-antibiotic ("post-abx") cohort showed the largest bar for Enterococcus. In the imipenem + P2A (1 mg) group, the pre-antibiotic ("pre-abx") cohort showed the largest bar for Clostridium and Lactobacillus, while the post-antibiotic ("post-abx") cohort showed the largest bar for Enterococcus. In the imipenem + P2A (10 mg) group, the pre-antibiotic ("pre-abx") cohort shows the largest bar for Lactobacillus, and the post-antibiotic ("post-abx") cohort shows the largest bar for Clostridium. In the imipenem + P2A (50 mg) group, the pre-antibiotic ("pre-abx") cohort shows the largest bar for Lactobacillus, and the post-antibiotic ("post-abx") cohort shows the largest bars for Lactobacillus and Clostridium. [Figure 9] Figure 1 shows the relative abundance of Enterococcus faecium in the feces of mice treated with imipenem plus cilastatin alone or with P2A at doses of 1 mg / kg, 10 mg / kg, and 50 mg / kg. [Figure 10] This figure shows the results of a survival study in which P2A was administered, with the endpoint being 40 days after bone marrow transplantation. "BM" refers to the cohort that received bone marrow cells only. "BM+T" refers to the cohort that received bone marrow cells and splenic T lymphocytes. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates, in part, to compositions and methods for reducing the incidence and / or severity of various complications associated with intravenous administration of β-lactam antibiotics in allogeneic hematopoietic stem cell transplantation (allo-HCT), including, but not limited to, acute graft-versus-host disease (aGVHD) and vancomycin-resistant enterococci (VRE) colonization and / or VRE bloodstream infections. In certain aspects, the present invention provides methods for reducing the incidence and / or severity of complications associated with intravenous administration of β-lactam antibiotics to allo-HCT recipients by administering a β-lactamase agent described herein to a subject in need thereof. In various aspects, the subject is receiving or has been receiving β-lactam antibiotics intravenously (IV).

[0016] Another aspect contemplates a method of reducing the incidence and / or severity of aGVHD by administering a β-lactamase agent described herein to a subject in need thereof. Another aspect contemplates a method of preventing VRE colonization and / or VRE bloodstream infection by administering a β-lactamase agent described herein to a subject in need thereof. In various embodiments, the β-lactamase agent is released into one or more regions of the intestinal tract, where it maintains and / or regulates the bacterial load and prevents the loss of bacterial strains, thereby preserving microbiome diversity and preventing microbiome monodominance.

[0017] β-lactamase inhibitors In some aspects, the present invention relates to the use of one or more beta-lactamases to reduce the incidence and / or severity of complications, including but not limited to aGVHD and VRE colonization and / or VRE bloodstream infections, associated with, for example, intravenous administration of beta-lactam antibiotics to allo-HCT recipients.

[0018] As used herein, β-lactamase refers to an enzyme that hydrolyzes β-lactams. Hydrolysis of the amide bond in the β-lactam ring biologically inactivates the antibacterial agent. As used herein, class A β-lactamase (Ambler classification) refers to a serine β-lactamase in which hydrolysis of the β-lactamase is mediated by a serine in the activation site (usually the 70th amino acid in α-helix 2). Class A β-lactamases include, but are not limited to, Bacillus cereus, such as Len-1, SHV-1, TEM-1, PSE-3 / PSE-3, ROB-1, 5 / B type 1, 569 / H type 1, and 569 / H type 3; Bacillus anthrasis sp; Bacillus licheniformis, such as PenP; Bacillus weihenstephanensis; Bacillus clausii; Staphylococcus aureus; aureus), PC1, Sme-1, NmcA, IMI-type, PER-type, VEB-type, GES-type, KPC-type, CME-, and CTX-M-type β-lactamases.

[0019] In various embodiments, a β-lactamase useful in the present invention has the amino acid sequence of SEQ ID NO: 1 (i.e., "SYN-004," "ribaxamase," or "P3A" from WO2011 / 148041, the entire contents of which are incorporated herein by reference). This sequence may be mutated to generate β-lactamase derivatives that may be utilized by the methods of the present invention.

[0020] [ka]

[0021] In some embodiments, the β-lactamase comprises an amino acid sequence having about 60% or greater sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) to SEQ ID NO:1.

[0022] In some embodiments, SEQ ID NO: 1 may have a Met and / or Thr before the first residue of the sequence. In various embodiments, the Met may be truncated. As described herein, sequences containing a Met and / or Thr before the first residue may be mutated to generate β-lactamase derivatives. In some embodiments, a leading Thr may confer increased stability of the enzyme compared to other preceding amino acids (e.g., Lys). For example, such a residue may confer increased resistance to aminopeptidases.

[0023] Also provided herein is the nucleic acid sequence of SYN-004 as SEQ ID NO:2.

[0024] [ka]

[0025] In some embodiments, polynucleotides of the invention have about 60% or greater sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) to SEQ ID NO:2.

[0026] In some embodiments, the β-lactamase, e.g., SYN-004, has substantial ceftriaxone hydrolysis activity. In some embodiments, the β-lactamase, e.g., SYN-004, hydrolyzes ceftriaxone substantially more efficiently than the naturally occurring β-lactamase, P1A.

[0027] In exemplary embodiments, the β-lactamase has 60% or greater sequence identity to SEQ ID NO:1 and comprises an amino acid sequence according to Ambler's classification: a hydrophobic residue other than alanine (A) at position 232, a hydrophilic residue other than alanine (A) at position 237, a hydrophobic residue other than alanine (A) at position 238, a hydrophilic residue other than serine (S) at position 240, and a hydrophilic residue other than aspartic acid (D) at position 276. In some embodiments, the hydrophobic residue other than alanine (A) at position 232 is glycine (G). In some embodiments, the hydrophilic residue other than alanine (A) at position 237 is serine (S). In some embodiments, the hydrophobic residue other than alanine (A) at position 238 is glycine (G). In some embodiments, the hydrophilic residue other than serine (S) at position 240 is aspartic acid (D). In some embodiments, the hydrophilic residue other than aspartic acid (D) at position 276 is asparagine (N). In some embodiments, the β-lactamase comprises one or more of A232G, A237S, A238G, S240D, and D276N. In some embodiments, the β-lactamase comprises all of A232G, A237S, A238G, S240D, and D276N, and the sequence is SEQ ID NO:3, i.e., P4A. In some embodiments, the β-lactamase and / or pharmaceutical composition comprises an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:3.

[0028] [ka]

[0029] In some embodiments, a beta-lactamase polypeptide of the invention comprises an amino acid sequence having about 60% or greater sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) to SEQ ID NO:3.

[0030] SEQ ID NO:4 is derived from SEQ ID NO:3 and further includes the signal and QASKT amino acids (the coding region is underlined).

[0031] [ka]

[0032] In some embodiments, a beta-lactamase polypeptide of the invention comprises an amino acid sequence having about 60% or greater sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) to SEQ ID NO:4.

[0033] In some embodiments, the beta-lactamase and / or pharmaceutical composition comprises an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:4.

[0034] An exemplary polypeptide of the invention is SEQ ID NO:5, which is the complete nucleotide sequence of the A232G, A237S, A238G, S240D, and D276N mutations, a Hind III site (AAGCTT in bold), and additional K and T amino acids. In some embodiments, the underlined portion of SEQ ID NO:5 is omitted and removed. The leader and additional nucleotides (the Hind III site and the K and T amino acids for the addition of the amino acid sequence QASKT) are underlined.

[0035] [ka]

[0036] In some embodiments, polynucleotides of the invention have about 60% or greater (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO:5 (with or without the underlined portion).

[0037] In various embodiments, the β-lactamase polypeptide has the sequence of SEQ ID NO:6 (ie, P2A) or is derived from one or more mutations of SEQ ID NO:6.

[0038] [ka]

[0039] In some embodiments, a beta-lactamase polypeptide of the invention comprises an amino acid sequence having about 60% or greater sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) to SEQ ID NO:6.

[0040] In some embodiments, the beta-lactamase and / or pharmaceutical composition comprises an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:6.

[0041] Additional sequences of β-lactamases, including P1A (i.e., SEQ ID NO: 1 except that position 276 is D, not N), P2A, P3A / SYN-004, and P4A, and derivatives thereof, are described, for example, in WO2011 / 148041 and PCT / US2015 / 026457, the entire contents of which are incorporated herein by reference.

[0042] Additionally, the β-lactamase polypeptide may include additional residues upstream from the first residue of SEQ ID NO:1 (see, e.g., JBC 258(18):11211, 1983, which includes the exolarge and exosmall forms of penP and penP1, the entire contents of which are incorporated herein by reference). Additionally, the β-lactamase polypeptide may also include additional residues downstream from the last residue of SEQ ID NO:1.

[0043] In some embodiments, the beta-lactamase comprises one or more (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10) mutations relative to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the beta-lactamase comprises a variant of SYN-004, e.g., a sequence having 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, 99.9% or more identity to SEQ ID NO:1 or SEQ ID NO:2. In various embodiments, one or more amino acids of SEQ ID NO: 1 are hydrophilic amino acids (e.g., polar, positively charged hydrophilic amino acids such as arginine (R) or lysine (K); polar, neutrally charged hydrophilic amino acids such as asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); polar, negatively charged hydrophilic amino acids such as aspartic acid (D) or glutamic acid (E), or aromatic, polar, positively charged hydrophilic amino acids such as histidine (H)), or hydrophobic amino acids (e.g., hydrophobic aliphatic amino acids such as glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V); hydrophobic aromatic amino acids such as phenylalanine (F), tryptophan (W), or tyrosine (Y)), or non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, Substitutions include naturally occurring amino acids such as β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids (e.g., β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids), and common amino acid analogs. In some embodiments, SEQ ID NO: 1 may have Met and / or Thr before the first residue of the sequence.These residues may be mutated in the same manner as above.

[0044] Exemplary mutations include the following: [Table 1]

[0045] In all of these mutants, the residue numbers correspond to SEQ ID NO: 1. These residue numbers may be converted to Ambler numbers (Ambler et al., 1991, "A standard numbering scheme for the Class A β-lactamases", Biochem. J. 276:269-272, the contents of which are incorporated herein by reference) through the use of any conventional bioinformatics method, for example, by using BLAST (Basic Local Alignment Search Tools) or FASTA (FAST-AII).

[0046] In various embodiments, the β-lactamase used in the present invention is produced in bacterial cells, such as E. coli cells (see, eg, PCT / US15 / 47187, the entire contents of which are incorporated herein by reference).

[0047] In various embodiments, the β-lactamases of the invention hydrolyze β-lactams. Hydrolysis of the amide bond in the β-lactam ring biologically inactivates the antibacterial agent. Examples of β-lactam antibiotics will be apparent to those skilled in the art. Illustrative examples of intravenous β-lactam antibiotics include, but are not limited to, penicillins (piperacillin / tazobactam), cephalosporins (cefepime), and / or carbapenems (meropenem; imipenem / cilastatin).

[0048] Treatment method Some embodiments provide compositions and methods that reduce the incidence and / or severity of complications associated with intravenous administration of β-lactam antibiotics to allo-HCT recipients, including, but not limited to, aGVHD and / or VRE colonization and / or VRE bloodstream infection, and / or infection or colonization, widespread colonization, or mono-dominance by one or more multidrug-resistant pathogens.

[0049] In some embodiments, monodominance includes about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% of the bacterial flora within a subject's microbiome being a single organism.

[0050] In certain embodiments, the present invention provides compositions and methods for preventing VRE colonization and / or VRE bloodstream infection, hi some embodiments, the methods of the present invention prevent intestinal dysbiosis associated with VRE colonization and / or VRE bloodstream infection.

[0051] In some embodiments, methods are provided for preventing VRE colonization and / or VRE bloodstream infection in transplant recipients comprising administering a β-lactamase of the invention.

[0052] In some aspects, methods are provided for preventing colonization and / or bloodstream infection with one or more multidrug-resistant organisms in a transplant recipient, comprising administering a β-lactamase of the invention. In some embodiments, the methods of the invention prevent intestinal dysbiosis associated with colonization and / or bloodstream infection with one or more multidrug-resistant organisms.

[0053] For example, in various embodiments, the present methods are directed to the prevention and treatment of the following pathogenic bacteria: Aeromonas hydrophila, Bacillus (e.g., Bacillus cereus), Bifidobacterium, Bordetella, Borrelia, Brucella, Burkholderia, C. difficile, Campylobacter (e.g., Campylobacter fetus, and Campylobacter jejuni). jejuni), Chlamydia, Chlamydophila, Clostridium (e.g., Clostridium botulinum, Clostridioides difficile (formerly Clostridium difficile)), and Clostridium perfringensperfringens), Corynebacterium, Coxiella, Ehrlichia, Enterobacteriaceae (e.g., carbapenem-resistant Enterobacteriaceae (CRE) and extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E)), fluoroquinolone-resistant Enterobacteriaceae, Enterococcus (e.g., vancomycin-resistant Enterococcus spp., extended-spectrum β-lactam-resistant Enterococci (ESBL-E)), , and vancomycin-resistant enterococci (VRE)), Escherichia (e.g., enteroaggregative E. coli, enterohemorrhagic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterotoxigenic E. coli (e.g., but not limited to, LT and / or ST), E. coli 0157:1-17, and multidrug-resistant E. coli), Francisella, Haemophilus, Helicobacter (e.g., Helicobacter pylori), pylori), Klebsiella (e.g., Klebsiellia pneumoniae and multidrug-resistant Klebsiella), Legionella, Leptospira, Listeria (e.g., Lysteria monocytogenes), Morganella, Mycobacterium, Mycoplasma, Neisseria, Orientia, Plesiomonas shigelloides shigelloides, antibiotic-resistant Proteobacteria, Proteus, Pseudomonas, Rickettsia, Salmonella (e.g., Salmonella paratyphi, Salmonella spp., and Salmonella typhi),typhi), Shigella (e.g., Shigella spp.), Staphylococcus (e.g., Staphylococcus aureus and Staphylococcus spp.), Streptococcus, Treponema, Vibrio (e.g., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio spp., and Vibrio vulnificus), and Yersinia (e.g., Yersinia enterocolitica).

[0054] In various embodiments, the methods are used in the treatment or prevention of infections caused by antibiotic-resistant bacteria, such as antibiotic-resistant Proteobacteria, VRE, carbapenem-resistant Enterobacteriaceae (CRE), fluoroquinolone-resistant Enterobacteriaceae, and extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E).

[0055] In certain embodiments, the present invention provides compositions and methods for reducing the incidence and / or severity of aGVHD. In various embodiments, the subject at risk of developing aGVHD is a transplant recipient. In other embodiments, the subject is an allo-HCT recipient. In some embodiments, the subject is a recipient of one or more of bone marrow cells, peripheral blood cells, and umbilical cord blood cells. In various embodiments, the subject at risk of developing aGVHD is receiving or has been receiving intravenously administered β-lactam antibiotics.

[0056] In some embodiments, the disclosed methods prevent intestinal dysbiosis associated with aGVHD. In various embodiments, the disclosed methods prevent a loss of gut microbiota diversity associated with aGVHD. For example, the disclosed methods contemplate administering a composition that modulates and / or reduces the monodominance of any given bacterial strain (e.g., Enterococcus spp.) associated with aGVHD. Other embodiments of the invention provide compositions and methods for preventing a loss of microbiome diversity in transplanted subjects.

[0057] In some embodiments, methods are provided for reducing the incidence and / or severity of aGVHD in transplant recipients, comprising administering a β-lactamase agent of the invention. For example, reducing the severity of aGVHD can include reducing the grade of aGVHD in a patient receiving the β-lactamase agent to less than Grade IV, which is classified as the most severe form of aGVHD, or reducing aGVHD by one or more grades (e.g., from Grade III to Grade II).

[0058] In some embodiments, the methods relate to reducing the incidence and / or severity of aGVHD in transplant recipients. In embodiments, the transplant recipient is a cancer patient, e.g., a patient who has undergone or has undergone radiation or chemotherapy followed by allo-HCT. In embodiments, the transplant recipient has a cancer of the blood or bone marrow. In embodiments, the transplant recipient is a recipient of a hematopoietic stem cell transplant. In certain embodiments, the cancer is selected from leukemia, lymphoma, myeloma, and myelodysplasia. In certain embodiments, the cancer is selected from osteosarcoma, Ewing's tumor, chordoma, and chondrosarcoma.

[0059] aGVHD is a deterioration of cells or tissues transplanted from a donor to a recipient due to the recipient's immune system recognizing the cells or tissues as foreign. Therefore, because class I MHC is present on more cells in the body, it is most preferable to transplant cells and tissues from a person with the most compatible class I MHC profile, followed by the most compatible class II MHC profile. Thus, in most transplant recipients, aGVHD results from immune system activation against incompatible class II MHC molecules and other polymorphic proteins (minor histocompatibility antigens).

[0060] One option for treating cancers of the blood or bone marrow is to kill existing blood and bone marrow cells, for example by radiation or chemotherapy, and then transplant similar cells from a healthy donor, called allogeneic hematopoietic stem cell transplant (allo-HCT).

[0061] In embodiments, the present methods relate to acute and chronic forms of GVHD. Classical acute or fulminant disease (aGVHD) is usually observed within the first 100 days after transplantation and is a major challenge to transplant effectiveness due to its associated morbidity and mortality. Chronic graft-versus-host disease (cGVHD) traditionally occurs after 100 days. The onset of moderate to severe cGVHD symptoms adversely affects long-term survival. After bone marrow transplantation, T cells present in the graft attack the transplant recipient's tissues after recognizing the host's tissues as antigenically foreign, either as contaminants or as tissues intentionally introduced into the host. T cells produce excessive amounts of cytokines, including TNFα and interferon-γ (IFNγ). Tissue damage in cGVHD is primarily due to fibrosis. A wide range of host antigens, particularly human leukocyte antigens (HLA), can cause GVHD. However, GVHD can occur even when the donor is an HLA-identical sibling. aGVHD is characterized by selective damage to the liver, skin, mucous membranes, and GI tract. Tissue damage in aGVHD is primarily due to apoptosis. The severity of aGVHD is graded from Scale 1 (mild) to Scale IV (very severe) based on the extent and type of lesions / rash (skin), the amount of diarrhea (GI), and serum pyruvate levels (liver). cGVHD is characterized by a more widespread tissue distribution than aGVHD. Skin and lungs, along with the GI tract, liver, eyes, musculoskeletal system, and hematopoietic system, are considered to be the primary targets of cGVHD. Hyperacute and acutely fatal forms of aGVHD can occur within the first 2 weeks of allogeneic HCT, usually due to significant HLA incompatibility or inadequate GVHD prophylaxis. Risk factors associated with cGVHD usually remain unchanged after prior adjustment for aGVHD, suggesting that cGVHD is not a simple evolution of the preceding aGVHD.

[0062] In embodiments, the disclosed methods relate to reducing the incidence and / or severity of aGVHD. In embodiments, the disclosed methods allow for the administration of β-lactamase to patients who have been treated with intravenous β-lactam antibiotics and who have one or more risk factors for aGVHD, such as HLA "mismatch" or unrelated donors, advanced patient age, female donor versus male recipient, intensity of conditioning regimen or intensity of total body irradiation during conditioning regimen, and donor lymphocyte infusion. In embodiments, the disclosed methods reduce the incidence and / or severity of symptoms of aGVHD, such as skin rash, GI tract disease, and liver symptoms.

[0063] In embodiments, the methods relate to GVHD defined by one or more of the Billingham criteria: 1) administration of an immunocompetent graft with viable and functional immune cells; 2) the recipient is immunologically histoincompatible; and 3) the recipient is immunocompromised, thereby failing to destroy or inactivate the transplanted cells.

[0064] Formulation / modified release profile In various embodiments, the invention utilizes modified-release formulations comprising at least one β-lactamase, which release a sufficient amount of β-lactamase into one or more regions of the GI tract. In some embodiments, the β-lactamase is SYN-004 (also known as P3A), or other β-lactamase agents described herein, or variants thereof (e.g., as described above). In some embodiments, the β-lactamase is SYN-006 (also known as P2A) or a variant thereof. For example, the formulation may release about 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006) into one or more regions of the GI tract after the stomach.

[0065] In various embodiments, the modified-release formulations of the present invention are designed for immediate release (e.g., upon ingestion). In various embodiments, the modified-release formulations may have a sustained-release profile, i.e., slow release of the active ingredient in the body (e.g., the GI tract) over an extended period of time. In various embodiments, the modified-release formulations may have a delayed-release profile, i.e., the active ingredient is not released immediately upon ingestion, but rather release of the active ingredient is delayed until the composition is depleted in the GI tract, for example, for release in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) or large intestine (e.g., one or more of the cecum, ascending, transverse, descending, or sigmoid portion of the colon, and rectum). For example, the composition may be enterically coated to delay release of the active ingredient until it reaches the small intestine or large intestine. In some embodiments, there is no substantial amount of the active ingredient of the present formulation in the feces.

[0066] In various embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) into one or more regions of the intestinal tract after the stomach. For example, the modified release formulations release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the intestinal tract. or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0067] In various embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the small intestine. For example, the modified release formulations release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the small intestine. or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0068] In some embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the duodenum. For example, the modified release formulations may release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, 117 % or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0069] In some embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof) in the jejunum. For example, the modified release formulations release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof) in the jejunum. or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0070] In certain embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the ileum / ileocecal region. For example, the modified release formulations may release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, 117% or releases 3% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0071] In various embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the large intestine. For example, the modified release formulations release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the large intestine. or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0072] In some embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof) in the cecum. For example, the modified release formulations may release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof) in the cecum. or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0073] In some embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the ascending portion of the colon. For example, the modified release formulation may release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, 117% or more, 1 releases 3% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0074] In some embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the transverse portion of the colon. For example, the modified release formulation may release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, 117% or more, 1 releases 3% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0075] In certain embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the descending portion of the colon. For example, the modified release formulation may release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, 117% or more, 1 releases 3% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0076] In certain embodiments, the modified release formulations of the present invention release 60% or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) in the sigmoid colon. For example, the modified release formulations may release 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, % or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0077] In various embodiments, the modified release formulation does not release substantially any beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof) in the stomach.

[0078] In some embodiments, the modified-release formulation releases β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) at a specific pH. For example, in some embodiments, the modified-release formulation is substantially stable in acidic environments and unstable (e.g., rapidly degrades or is physically unstable) in near-neutral to alkaline environments. In some embodiments, stability indicates substantially no release, and instability indicates substantial release. For example, in some embodiments, the modified-release formulation is substantially stable at a pH of about 7.0 or less, about 6.5 or less, about 6.0 or less, about 5.5 or less, about 5.0 or less, about 4.5 or less, about 4.0 or less, about 3.5 or less, about 3.0 or less, about 2.5 or less, about 2.0 or less, about 1.5 or less, or about 1.0 or less. In some embodiments, the formulation is stable at low pH, and therefore, substantially no release occurs, for example, in the stomach. In some embodiments, the modified release formulation is substantially stable at a pH of about 1 to about 4 or less and substantially unstable at higher pH values. In these embodiments, the modified release formulation is not substantially released in the stomach. In these embodiments, the modified release formulation is substantially released in the small intestine (e.g., one or more of the duodenum, jejunum, ileum) and / or large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon). In some embodiments, the modified release formulation is substantially stable at a pH of about 4 to about 5 or less and, therefore, is substantially unstable at higher pH values ​​and is therefore not substantially released in the stomach and / or small intestine (e.g., one or more of the duodenum, jejunum, ileum). In these embodiments, the modified release formulation is substantially released in the large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon). In various embodiments, the pH values ​​described herein may be adjusted as known in the art to account for the patient's state, e.g., fasting or postprandial.

[0079] In some embodiments, the modified release formulation is substantially stable in gastric fluids and substantially unstable in intestinal fluids, and therefore is substantially released in the small intestine (e.g., one or more of the duodenum, jejunum, ileum) and / or large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon).

[0080] In some embodiments, the modified-release formulations are stable in gastric fluid or stable in an acidic environment, releasing about 30% or less by weight of the beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof) and / or additional therapeutic agent in gastric fluid having a pH of about 4 to about 5 or less or simulated gastric fluid having a pH of about 4 to about 5 or less in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes. The modified-release formulations of the present invention may release about 0% to about 30% by weight, about 0% to about 25% by weight, about 0% to about 20% by weight, about 0% to about 15% by weight, about 0% to about 10% by weight, about 5% to about 30% by weight, about 5% to about 25% by weight, about 5% to about 20% by weight, about 5% to about 15% by weight, or about 5% to about 10% by weight of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof) and / or additional therapeutic agent in gastric fluid having a pH of about 4 to about 5 or less, or in simulated gastric fluid having a pH of about 4 to about 5 or less, in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes. The modified-release formulations of the invention may release about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight of the total beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein and variants thereof) and / or additional therapeutic agent in gastric fluid having a pH of 5 or less or simulated gastric fluid having a pH of 5 or less in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes.

[0081] In some embodiments, the modified-release formulations are unstable in intestinal fluids. These modified-release formulations release about 70% or more by weight of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) and / or additional therapeutic agent in the modified-release formulation into intestinal fluid or simulated intestinal fluid in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes. In some embodiments, the modified-release formulations are unstable in near-neutral to alkaline environments. These modified-release formulations release about 70% or more by weight of the beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof) and / or additional therapeutic agent in intestinal fluid having a pH of about 4 to about 5 or higher or simulated intestinal fluid having a pH of about 4 to about 5 or higher in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes. A modified-release formulation that is unstable in a near-neutral or alkaline environment may be soluble in a liquid (e.g., a liquid having a pH of greater than about 5) for about 5 to about 90 minutes, about 10 to about 90 minutes, about 15 to about 90 minutes, about 20 to about 90 minutes, about 25 to about 90 minutes, about 30 to about 90 minutes, about 5 to about 60 minutes, about 10 to about 60 minutes, about 15 to about 60 minutes, about 20 to about 60 minutes, about 25 to about 90 minutes, or about 30 to about 60 minutes. For example, 70% by weight or more of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) and / or additional therapeutic agent may be released into a liquid having a pH of about 5 to about 14, about 6 to about 14, about 7 to about 14, about 8 to about 14, about 9 to about 14, about 10 to about 14, or about 11 to about 14.

[0082] Examples of simulated gastric and intestinal fluids include, but are not limited to, those disclosed on page 2858 of 2005 Pharmacopeia 23NF / 28USP in Test Solutions, and / or other simulated gastric and intestinal fluids known to those skilled in the art, such as simulated gastric and / or intestinal fluids prepared without the use of enzymes.

[0083] In some embodiments, the modified-release formulation may remain essentially intact or may be essentially insoluble in gastric fluid. The modified-release formulation may include one or more pH-dependent delayed-release coatings. The pH-dependent delayed-release coating may be substantially stable in acidic environments (pH of about 5 or less) and substantially unstable in near-neutral to alkaline environments (pH above about 5). For example, the delayed-release coating may essentially disintegrate or dissolve in near-neutral to alkaline environments such as those found in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) and / or large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon).

[0084] Alternatively, the stability of the modified-release formulation may be enzyme-dependent. In such embodiments, the modified-release formulation may include one or more enzyme-dependent delayed-release coatings. An enzyme-dependent delayed-release coating may be substantially stable in a liquid that does not contain a particular enzyme and substantially unstable in a liquid that contains the particular enzyme. The delayed-release coating will essentially disintegrate or dissolve in a liquid that contains the appropriate enzyme. Enzyme-dependent control can be achieved, for example, by using a material, such as galactomannan, that releases the active ingredient only when exposed to enzymes in the intestinal tract. The stability of the modified-release formulation may also depend on the stability of the enzyme in the presence of microbial enzymes present in the intestinal flora.

[0085] In various embodiments, modified-release formulations comprising a β-lactamase (e.g., SYN-004 or SYN-006, or a variant thereof) are substantially stable in chyme, e.g., in some embodiments, lose no more than about 50%, 40%, 30%, 20%, or 10% of β-lactamase activity at about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour after administration.

[0086] In some embodiments, dual pulse formulations are provided. In various embodiments, the present invention provides modified-release formulations that release multiple doses of β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) at different locations along the intestinal tract, at different times, and / or at different pHs. In an exemplary embodiment, the modified-release formulation comprises a first dose of β-lactamase and a second dose of β-lactamase, where the first and second doses are released at different locations along the intestinal tract, at different times, and / or at different pHs. For example, the first dose is released in the duodenum and the second dose is released in the ileum. In another embodiment, the first dose is released in the jejunum and the second dose is released in the ileum. In another embodiment, the first dose is released at a location along the small intestine (e.g., the duodenum), while the second dose is released at a location along the large intestine (e.g., the ascending colon). In various embodiments, the modified release formulation may release one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more doses of beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof) at different locations along the intestine, at different times, and / or at different pHs. Furthermore, the description of dual-pulse formulations herein applies to modified release formulations that release a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof) and an additional therapeutic agent.

[0087] In various embodiments, the present invention employs modified-release formulations of a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof), which may further comprise a pharmaceutically acceptable carrier or non-medicinal excipient. As one of skill in the art will recognize, the formulation may be in any suitable form appropriate for the desired use and route of administration.

[0088] In some embodiments, the modified release formulation comprising the β-lactamase (and / or additional therapeutic agent) is administered orally, intravenously, or parenterally. In some embodiments, the modified release formulation comprising the β-lactamase (and / or additional agent) is not administered intravenously, e.g., to prevent interference with systemically administered antibiotics. In other embodiments, the route of administration includes, e.g., oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation, or topical administration, particularly to the ear, nose, eye, or skin.

[0089] Any modified-release formulation containing a β-lactamase (and / or additional therapeutic agent) as described herein may be administered orally. Such inventive formulations may also be administered by other conventional routes, for example, by intravenous infusion or intravenous bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), or may be administered in conjunction with additional therapeutic agents. Administration may be systemic or local. In some embodiments, administration is not at the site of infection, for example, to avoid hydrolysis of the antibiotic at the site of infection. Various delivery systems are known and may be used for administration, for example, encapsulation in liposomes, microparticles, microcapsules, capsules, etc. In specific embodiments, local administration to the area requiring treatment may be desirable.

[0090] Suitable dosage forms for oral use include, for example, solid dosage forms such as tablets, dispersible powders, granules, and capsules. In one embodiment, the modified release formulation is in the form of a capsule. In another embodiment, the modified release formulation is in the form of a tablet. In yet another embodiment, the modified release formulation is in the form of a softgel capsule. In a further embodiment, the modified release formulation is in the form of a gelatin capsule or a hydroxypropylmethylcellulose (HPMC) capsule.

[0091] In some dosage forms, the agents described herein may be combined with at least one inert, pharmaceutically acceptable excipient or carrier (such as sodium citrate, dicalcium phosphate, etc.), and / or a) excipients or fillers (such as starch, lactose, sucrose, glucose, mannitol, silicic acid, microcrystalline cellulose, and Baker's Special Sugar), b) binders (such as carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, acacia, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropyl cellulose (HPC), and hydroxymethylcellulose), c) humectants (such as glycerol), d) disintegrants (such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, sodium carbonate, crospovidone (cross-linked polyvinylpyrrolidone), croscarmellose sodium (cross-linked carboxymethylcellulose), or e) agar-agar, calcium carbonate, d ... The non-medicinal ingredients may be mixed with crosslinked polymers such as sodium hydroxymethylcellulose, sodium starch glycolate, etc., e) solution retarders (such as paraffin), f) absorption enhancers (such as quaternary ammonium compounds), g) wetting agents (such as cetyl alcohol and glycerol monoacetate), h) absorbents (such as kaolin and bentonite clay), and i) lubricants (such as talc, calcium stearate, magnesium stearate, sodium polyethylene glycol, sodium lauryl sulfate, glyceryl behenate, etc.), and mixtures of such non-medicinal ingredients. Those skilled in the art will appreciate that a particular non-medicinal ingredient may have more than one function in an oral dosage form. For example, in the case of oral dosage forms such as capsules or tablets, the dosage form may also include a buffering agent.

[0092] The modified release formulation may further comprise a surfactant.Surfactants suitable for use in the present invention include, but are not limited to, any pharmaceutically acceptable non-toxic surfactant.Surfactants suitable for use in the present invention include, but are not limited to, polyethoxylated fatty acids, PEG-fatty acid diesters, mixtures of PEG-fatty acid monoesters and PEG-fatty acid diesters, polyethylene glycol glycerol fatty acid esters, alcohol-oil transesterification products, polyglycerinated fatty acids, propylene glycol fatty acid esters, mixtures of propylene glycol esters and glycerol esters, monoglycerides and diglycerides, sterols and sterol derivatives, propylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl esters, sugar esters, polyethylene glycol alkylphenols, polyoxyethylene-polyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, ionic surfactants, and mixtures thereof. In some embodiments, the compositions of the present invention may include one or more surfactants, including, but not limited to, sodium lauryl sulfate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and triethyl citrate.

[0093] The modified-release formulations may also include pharmaceutically 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.

[0094] The modified release formulation may also include one or more coating solvents. For example, some of the more common solvents that may be used to apply the delayed-release coating composition include isopropyl alcohol, acetone, and methylene chloride.

[0095] The modified-release formulation may also contain one or more alkaline substances. Alkaline substances suitable for use in the present compositions include, but are not limited to, sodium, potassium, calcium, magnesium, and aluminum salts of acids such as phosphate compounds, carbonate compounds, citrate compounds, and other aluminum / magnesium compounds. Additionally, the alkaline substance may be selected from antacid materials such as aluminum hydroxide, calcium hydroxide, magnesium hydroxide, and magnesium oxide.

[0096] Solid oral dosage forms can be prepared, for example, by granulating (e.g., wet granulation or dry granulation) an agent of the invention with one or more suitable non-medicinal ingredients. Alternatively, an agent of the invention can be layered onto inert cores (e.g., nonpareil / sugar spheres, such as sucrose spheres, or silica spheres) using conventional methods such as fluidized bed coating or pan coating, or extruded and spheronized into active compound-containing tablets using methods well known in the art. In embodiments, a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and their variants) is spray-coated onto sucrose spheres. Such spheres can then be incorporated into tablets or capsules using conventional methods.

[0097] In addition to the active agent, suspensions may contain suspending agents such as, for example, ethoxylated isocetyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and the like, and mixtures thereof.

[0098] Besides inert diluents, the oral compositions may also include adjuvants such as sweetening, flavoring, and perfuming agents.

[0099] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, and emulsions. They may also be prepared in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injectable medium immediately before use. These may contain, for example, suspending agents or dispersing agents known in the art.

[0100] Formulations containing a β-lactamase (and / or additional therapeutic agent) may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agent into association with a carrier which constitutes one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier (e.g., phosphate-buffered saline), a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired dosage form (e.g., by wet or dry granulation, powder mixture, etc., followed by tabletting using conventional methods known in the art).

[0101] In various embodiments, the modified release formulations of the present invention may utilize one or more modified release coatings, such as a delayed release coating to provide effective, delayed yet substantial delivery of a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof), optionally along with other additional therapeutic agents, to the GI tract.

[0102] In some embodiments, the delayed-release coating comprises an enteric agent that is substantially stable in acidic environments and unstable in near-neutral to alkaline environments, hi some embodiments, the delayed-release coating comprises an enteric agent that is substantially stable in gastric fluids. Enteric coating agents can be selected from, for example, methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropyl cellulose phthalate, polyvinyl acetate phthalate, carboxymethyl ethyl cellulose, and EUDRAGIT®-type polymers (poly(methacrylic acid, methyl methacrylate), hydroxypropyl methylcellulose acetate succinate, cellulose acetate trimellitate, shellac, or solutions or dispersions of other enteric coating polymers. EUDRAGIT®-type polymers include, for example, EUDRAGIT® FS 30D, L 30 D-55, L 100-55, L 100, L 12.5, L 12.5 P, RL 30 D, RL PO, RL 100, RL 12.5, RS 30 D, RS PO, RS 100, RS 12.5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12.5, and S 12,5 P. Similar polymers include Kollicoat® MAE 30 DP and Kollicoat® MAE 100 P. In some embodiments, one or more of EUDRAGIT® FS 30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5 S 12,5 P, Kollicoat® MAE 30 DP, and Kollicoat® MAE 100 P are used. The following documents are incorporated by reference in their entirety:(1) Thakral et al., "Eudragit®: A technology evaluation," Expert Opinion on Drug Delivery, Vol. 10 (2013), pp. 131-149; (2) Niranjan Patra et al., "Pharmaceutical significance of Eudragit: A review," Future Journal of Pharmaceutical Sciences, Vol. 3 (10.1016 / j.fjps.2017.02.001); (3) Sonje, Abhijit and Chandra, Amrish, "Comprehensive review on eudragit polymers," International Research Journal of Pharmacy, Vol. 4 (10.7897 / 2230-8407.04515). In various embodiments, the enteric coating may be a combination of the aforementioned solutions or dispersions. In one embodiment, the delayed-release coating comprises the enteric coating EUDRAGIT® L 30 D-55.

[0103] In some embodiments, one or more coating system additives may be used with the enteric coating. For example, one or more PlasACRYL™ additives may be used as anti-adhesive coating additives. Exemplary PlasACRYL™ additives include, but are not limited to, PlasACRYL™ HTP20 and PlasACRYL™ T20. In some embodiments, PlasACRYL™ HTP20 is formulated with a EUDRAGIT® L 30 D-55 coating. In another embodiment, PlasACRYL™ T20 is formulated with a EUDRAGIT® FS 30 D coating.

[0104] In another embodiment, the delayed-release coating can degrade over time in aqueous solution, regardless of pH and / or the presence of enzymes in the solution. Such coatings can include water-insoluble polymers. Therefore, their solubility in aqueous solution is pH-independent. The term "pH-independent" as used herein means that the water permeability of the polymer and its ability to release the pharmaceutical ingredient are not dependent on pH and / or only slightly dependent on pH. Such coatings can be used, for example, to prepare sustained-release formulations. Suitable water-insoluble polymers include pharmaceutically acceptable, non-toxic polymers that are substantially insoluble in aqueous media (e.g., water) independently of the pH of the solution. Suitable polymers include, but are not limited to, cellulose ethers, cellulose esters, or cellulose ether esters, i.e., cellulose derivatives in which some of the hydroxy groups on the cellulose backbone are replaced with alkyl groups and some are modified with alkanoyl groups. Examples include ethyl cellulose, acetyl cellulose, and nitrocellulose. Other examples of insoluble polymers include, but are not limited to, lacquers, acrylic and / or methacrylic ester polymers, acrylate or methacrylate polymers or copolymers with low quaternary amine content, or mixtures thereof. Other examples of insoluble polymers include EUDRAGIT RS®, EUDRAGIT RL®, and EUDRAGIT NE®. Insoluble polymers useful in the present invention include polyvinyl esters, polyvinyl acetates, polyacrylic esters, and butadiene styrene copolymers. In some embodiments, colonic delivery is achieved using a slowly-eroding wax plug (e.g., various PEGs, including PEG 6000).

[0105] In other embodiments, the delayed-release coating can be degraded by microbial enzymes present in the intestinal flora. In one embodiment, the delayed-release coating can be degraded by bacteria present in the small intestine. In another embodiment, the delayed-release coating can be degraded by bacteria present in the large intestine.

[0106] In various embodiments, the present invention provides a formulation comprising a core particle having a base coat containing one or more beta-lactamases (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof), and a delayed-release coating disposed on the coated core particle. The delayed-release coating is substantially stable in acidic environments and / or gastric fluids, and / or is unstable in near-neutral to alkaline environments or in intestinal fluids, such that the coated core particle is exposed to intestinal fluids. The base coat containing one or more beta-lactamases may further comprise one or more additional therapeutic agents. Optionally, multiple base coats may be applied to the core, each of which may contain a beta-lactamase and / or additional therapeutic agent. In some embodiments, the core particle may comprise sucrose. The formulations may be prepared by methods known in the art. For example, a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) can be sprayed onto an inert core (e.g., a sucrose core or sucrose sphere) and spray-dried with an enteric layer (EUDRAGIT L30 D-55) to form a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof)-containing pellet.

[0107] Optionally, the core particle may contain one or more beta-lactamases (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof) and / or one or more additional therapeutic agents. In some embodiments, one or more doses of beta-lactamase may be encapsulated in the core particle, e.g., in the form of microspheres. For example, the beta-lactamase may be combined with a polymer (e.g., latex) and then formed into a particulate microencapsulated enzyme formulation without the use of a sucrose core. The formed microspheres may then optionally be coated with a delayed-release coating.

[0108] Various approaches are known for producing particles (e.g., microspheres, aggregates, etc.) suitable for encapsulating enzymes. They typically contain two or more phases: an enzyme-containing phase and a polymer-containing phase that forms the backbone of the microparticles. The most common are coacervates, in which the polymer separates from the solvent phase upon addition of a third component, or multiple emulsions, such as water-in-oil-in-water (w / o / w) emulsions, in which an inner aqueous phase contains the protein, a middle organic phase contains the polymer, and an outer aqueous phase contains a stabilizer that supports the w / o / w double emulsion until the solvent is removed to form the microspheres. Alternatively, a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and their variants) and a stabilizing non-active ingredient (e.g., trehalose, mannitol, Tween 80, polyvinyl alcohol) are combined, sprayed from an aqueous solution, and recovered. The particles are then suspended in a dry, water-immiscible organic solvent containing the polymer and release-modifying compound, and the suspension is then sonicated to disperse the particles. Other approaches use an aqueous phase but no organic solvent. Specifically, enzymes, buffering components, polymer latex, and stabilizing and release-modifying non-active ingredients are dissolved / dispersed in water. The aqueous dispersion is spray-dried to cause latex coalescence, entrapping the protein and non-active ingredients in coalesced latex particles. If the release-modifying agent is insoluble under acidic conditions but soluble at higher pH (e.g., carboxylic acids), release from the matrix is ​​inhibited in the gastric environment.

[0109] In some embodiments, prior to applying the delayed-release coating to the coated core particles, the particles may optionally be coated with one or more separating layers comprising non-medicinal pharmaceutical ingredients, including, for example, alkaline compounds such as pH buffering compounds, which essentially separate the coated core particles from the delayed-release coating.

[0110] The separating layer can be applied to the coated core particles by a coating or layering process using a coating device such as a coating pan or a coating granulator, or in a fluidized bed device using water and / or an organic solvent for the coating process. Alternatively, the separating layer can be applied to the core material using a powder coating technique. Materials for the separating layer include pharmaceutically acceptable compounds, such as sugars, polyethylene glycols, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxymethylpropyl cellulose, sodium carboxymethyl cellulose, and the like, used alone or in mixtures. Additives such as plasticizers, colorants, pigments, excipients, anti-adhesives, and anti-static agents, such as magnesium stearate, titanium dioxide, talc, and other additives, can also be included in the separating layer.

[0111] In some embodiments, coated particles having a delayed-release coating may be further coated with a topcoat layer. The topcoat layer may be applied as described for other coating compositions. Topcoat materials are pharmaceutically acceptable compounds, such as sugars, polyethylene glycols, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and sodium carboxymethyl cellulose, used alone or in mixtures. Topcoat materials may prevent potential aggregation of particles coated with the delayed-release coating, protect the delayed-release coating from cracking during compression, or facilitate the tableting process.

[0112] In various embodiments, the formulation may comprise a plurality of modified-release particles, modified-release pellets, or modified-release microspheres. In some embodiments, the formulation is in the form of a capsule comprising a plurality of pellets. In some embodiments, the formulation is in the form of a capsule comprising a plurality of microspheres.

[0113] In some embodiments, the modified-release formulation may be a capsule filled with multiple β-lactamase-containing pellets (e.g., pellets containing SYN-004 or SYN-006 (or other β-lactamase agents described herein and variants thereof)) from which the β-lactamase is released. In certain embodiments, the capsule is a gelatin capsule, such as a hard gelatin capsule. In other embodiments, the capsule is a hydroxypropyl methylcellulose (HPMC) capsule. For example, the formulation may be in the form of a capsule containing multiple pellets. For example, the formulation may be in the form of a capsule, such as a gelatin capsule or a hydroxypropyl methylcellulose (HPMC) capsule, containing multiple enteric-coated pellets containing β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof). In such embodiments, a combination of pellets may be utilized, each designed to be released at a specific time or location. In various embodiments, the pellets (e.g., enteric-coated pellets) are designed to release the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) into one or more regions of the intestinal tract after passing through the stomach unchanged. In some embodiments, the β-lactamase-containing pellets may be enteric-coated to release the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) at different pH values ​​in the intestinal tract.

[0114] In various embodiments, the formulations of the invention may be in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing a plurality of enteric-coated β-lactamase-containing pellets. In such embodiments, the pellets (or individual pellets) comprise sucrose spheres sprayed with a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof), e.g., a β-lactamase such as SYN-004 or SYN-006 or variants thereof, a binder non-medicinal ingredient (e.g., hydroxypropyl cellulose (HPC)), an enteric polymer (e.g., EUDRAGIT L 30 D-55), a plasticizer (e.g., triethyl citrate), a glidant (e.g., glyceryl monostearate), an emulsifier, and a buffer salt.

[0115] In various embodiments, the formulations of the present invention may be in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing a plurality of enteric-coated β-lactamase-containing pellets. In such embodiments, the pellet (or individual pellet) contains about 10-20% by weight of the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof). For example, the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof) may be present at about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by weight. In some embodiments, the pellet (or individual pellets) comprise about 20-30% by weight of sucrose spheres, onto which a β-lactamase, such as SYN-004 or SYN-006 or a variant thereof, has been sprayed. For example, the sucrose spheres may be present at about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by weight. In various embodiments, the pellet (or individual pellets) comprise about 30-40% by weight of a binder non-medicinal ingredient (e.g., hydroxypropyl cellulose (HPC)). For example, the binder non-medicinal ingredient may be present at about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% by weight. In some embodiments, the pellet (or individual pellets) contain about 15-25% by weight of an enteric polymer (e.g., EUDRAGIT L 30 D-55). For example, the enteric polymer may be present at about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% by weight. In some embodiments, the pellet (or individual pellets) contain about 1.5-2.5% by weight of a plasticizer (e.g., triethyl citrate).For example, the plasticizer may be present at about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% by weight. In some embodiments, the pellet (or individual pellet) comprises about 0.5-1.5% by weight of a glidant (e.g., glyceryl monostearate). For example, the glidant may be present at about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, or about 1.5% by weight. In some embodiments, the pellets (or individual pellets) contain about 0.1-1.0% by weight of an emulsifier (e.g., polysorbate-80). For example, the emulsifier may be present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% by weight. In some embodiments, the pellets (or individual pellets) further contain about 1-2% by weight of a buffer salt. For example, the buffer salt may be present at about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2% by weight. Weights stated herein refer to the combined weight of all ingredients, excluding the weight of the capsule itself.

[0116] In some embodiments, the pellets (or individual pellets) comprise about 16% by weight of a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof); about 23% by weight of sucrose spheres; about 35% by weight of binder non-medicinal ingredients (e.g., hydroxypropyl cellulose (HPC)); about 21% by weight of an enteric polymer (e.g., EUDRAGIT L 30 D-55); about 2% by weight of a plasticizer (e.g., triethyl citrate); about 1% by weight of a glidant (e.g., glyceryl monostearate); about 0.5% by weight of an emulsifier (e.g., polysorbate-80); and about 2% by weight of a buffer salt. The weights listed herein refer to the combined weight of all components, excluding the weight of the capsule itself.

[0117] For example, the pellets (or individual pellets) contain about 15.8% by weight of a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof); about 23.3% by weight of sucrose spheres; about 35% by weight of binder non-medicinal ingredients (e.g., hydroxypropyl cellulose (HPC)); about 20.8% by weight of an enteric polymer (e.g., EUDRAGIT L 30 D-55); about 2.1% by weight of a plasticizer (e.g., triethyl citrate); about 1.0% by weight of a glidant (e.g., glyceryl monostearate); about 0.4% by weight of an emulsifier (e.g., polysorbate-80); and about 1.6% by weight of a buffer salt. The weights listed herein refer to the combined weight of all components, excluding the weight of the capsule itself.

[0118] In various embodiments, the formulations of the invention are in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 75 mg of a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof). The capsule contains a plurality of enteric-coated β-lactamase-containing pellets. In such embodiments, the formulation contains about 10-20% by weight of a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof). For example, the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) may be present at about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% by weight. In some embodiments, the formulation comprises about 15-25% sucrose spheres by weight. For example, the sucrose spheres may be present at about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% by weight. In various embodiments, the formulation comprises about 25-35% by weight of a binder non-medicinal ingredient (e.g., hydroxypropyl cellulose (HPC)). For example, the binder non-medicinal ingredient may be present at about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% by weight. In some embodiments, the formulation comprises about 10-25% by weight of an enteric polymer (e.g., EUDRAGIT L 30 D-55). For example, the enteric polymer may be present at about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% by weight. In some embodiments, the formulation comprises about 1.5-2.5% by weight of a plasticizer (e.g., triethyl citrate).For example, the plasticizer may be present at about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% by weight. In some embodiments, the formulation includes about 0.5-1.5% by weight of a glidant (e.g., glyceryl monostearate). For example, the glidant may be present at about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, or about 1.5% by weight. In some embodiments, the formulation includes about 0.1-1.0% by weight of an emulsifier (e.g., polysorbate-80). For example, the emulsifier may be present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% by weight. In some embodiments, the formulation includes about 1-2% by weight of a buffer salt. For example, it may be present at about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2% by weight. In some embodiments, the formulation may include about 10-20% by weight of a gelatin capsule or HPMC capsule. For example, the gelatin or HPMC capsule may be about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% by weight.

[0119] In some embodiments, a formulation of the invention comprises about 75 mg of a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof). In such embodiments, the formulation comprises about 13% by weight of a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof); about 19% by weight of sucrose spheres; about 29% by weight of binder non-medicinal ingredients (e.g., hydroxypropyl cellulose (HPC)); about 17% by weight of an enteric polymer (e.g., EUDRAGIT L 30 D-55); about 2% by weight of a plasticizer (e.g., triethyl citrate); about 1% by weight of a glidant (e.g., glyceryl monostearate); about 0.5% by weight of an emulsifier (e.g., polysorbate-80); about 1% by weight of a buffer salt; and about 17% by weight of a gelatin capsule or HPMC capsule.

[0120] For example, the formulation may comprise about 13.1% by weight of a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof); about 19.4% by weight of sucrose spheres; about 29.1% by weight of binder non-medicinal ingredients (e.g., hydroxypropyl cellulose (HPC)); about 17.3% by weight of an enteric polymer (e.g., EUDRAGIT L 30 D-55); about 1.7% by weight of a plasticizer (e.g., triethyl citrate); about 0.9% by weight of a glidant (e.g., glyceryl monostearate); about 0.4% by weight of an emulsifier (e.g., polysorbate-80); about 1.3% by weight of a buffer salt; and about 16.8% by weight of a gelatin capsule or HPMC capsule.

[0121] In various embodiments, the formulations of the invention are in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 25 mg of a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof). The capsule contains a plurality of enteric-coated β-lactamase-containing pellets. In such embodiments, the formulation contains about 5-15% by weight of a β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof). For example, the β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) may be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% by weight. In some embodiments, the formulation comprises about 10-20% sucrose spheres by weight. For example, the sucrose spheres may be present at about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% by weight. In various embodiments, the formulation comprises about 15-25% by weight of a binder non-medicinal ingredient (e.g., hydroxypropyl cellulose (HPC)). For example, the binder non-medicinal ingredient may be present at about 15%, about 16%, about 1%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% by weight. In some embodiments, the formulation includes about 10-20% by weight of an enteric polymer (e.g., EUDRAGIT L 30 D-55). For example, the enteric polymer may be present at about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% by weight. In some embodiments, the formulation includes about 1.0-2.0% by weight of a plasticizer (e.g., triethyl citrate).For example, the plasticizer may be present at about 1.0 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, about 1.6 wt%, about 1.7 wt%, about 1.8 wt%, about 1.9 wt%, or about 2.0 wt%. In some embodiments, the formulation includes about 0.1-1.0 wt% of a glidant (e.g., glyceryl monostearate). For example, the glidant may be present at about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, or about 1 wt%. In some embodiments, the formulation includes about 0.1-1.0 wt% of an emulsifier (e.g., polysorbate-80). For example, the emulsifier may be present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% by weight. In some embodiments, the formulation includes about 0.5-1.5% by weight of a buffer salt. For example, the buffer salt may be present at 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, or about 1.5% by weight. In some embodiments, the formulation may include about 30-40% by weight of a gelatin capsule or HPMC capsule. For example, the gelatin or HPMC capsule may be 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% by weight.

[0122] In some embodiments, a formulation of the invention comprises about 25 mg of a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof). In such embodiments, the formulation comprises about 10% by weight of a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof); about 15% by weight of sucrose spheres; about 22% by weight of binder non-medicinal ingredients (e.g., hydroxypropyl cellulose (HPC)); about 13% by weight of an enteric polymer (e.g., EUDRAGIT L 30 D-55); about 1% by weight of a plasticizer (e.g., triethyl citrate); about 0.5% by weight of a glidant (e.g., glyceryl monostearate); about 0.3% by weight of an emulsifier (e.g., polysorbate-80); about 1% by weight of a buffer salt; and about 38% by weight of a gelatin capsule or HPMC capsule.

[0123] For example, the formulation may comprise about 9.8% by weight of a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof); about 14.5% by weight of sucrose spheres; about 21.8% by weight of binder non-medicinal ingredients (e.g., hydroxypropyl cellulose (HPC)); about 13% by weight of an enteric polymer (e.g., EUDRAGIT L 30 D-55); about 1.3% by weight of a plasticizer (e.g., triethyl citrate); about 0.6% by weight of a glidant (e.g., glyceryl monostearate); about 0.3% by weight of an emulsifier (e.g., polysorbate-80); about 1.0% by weight of a buffer salt; and about 37.7% by weight of a gelatin capsule or HPMC capsule.

[0124] The present invention also provides modified-release formulations that release multiple doses of β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) and / or additional therapeutic agents along the GI tract. In such embodiments, the overall release profile of such formulations may be tailored, for example, by utilizing multiple particle types or multiple layers. In certain embodiments, a first dose of β-lactamase may be formulated for release in, for example, the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) or the large intestine (e.g., one or more of the cecum, ascending, transverse, descending, or sigmoid portion of the colon, and the rectum), and a second dose is formulated for delayed release at a different location in, for example, the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) or the large intestine (e.g., one or more of the cecum, ascending, transverse, descending, or sigmoid portion of the colon, and the rectum). Alternatively, multiple doses may be released at different locations along the intestinal tract. For example, in one embodiment, a first dose of β-lactamase may be formulated for release, for example, in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum), and a second dose may be formulated for delayed release, for example, at another location in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum). In another embodiment, a first dose of β-lactamase may be formulated for release, for example, in the large intestine (e.g., one or more of the cecum, ascending, transverse, descending, or sigmoid portion of the colon, and rectum), and a second dose may be formulated for delayed release, for example, at another location in the large intestine (e.g., one or more of the cecum, ascending, transverse, descending, or sigmoid portion of the colon, and rectum).

[0125] In various embodiments, the agents described herein may be in the form of pharmaceutically acceptable salts, i.e., salts suitable for use in contact with the tissues of humans and other animals without excessive toxicity, irritation, allergic response, etc., and commensurate with a reasonable risk-benefit ratio. Pharmaceutically acceptable salts are well known in the art. Salts can be prepared in situ during the final isolation and purification of the therapeutic agent, or separately by reacting a free base function with a suitable acid or a free acid function with a suitable alkali moiety. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lanthanide, and lanthanide. Examples of suitable salts include butobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0126] In various embodiments, the present formulations offer numerous advantages. For example, the inventors have successfully formulated a protein (i.e., β-lactamase), which is itself challenging. This is further compounded by the GI tract environment in which the present formulations, in various embodiments, release the drug. In addition, in various embodiments, the present formulations provide a sufficiently slow GI tract release to allow for good coverage in the GI tract from the adverse effects of various antibiotics, e.g., in the small intestine (an advantage accentuated by the increased β-lactamase half-life commensurate with the slower release). Furthermore, by coating the drug substance layer of the present pellets with HPC rather than EUDRAGIT, for example, the present formulations minimize the amount of EUDRAGIT in the formulation, thereby reducing the potential for dose-limiting toxicity and manufacturing complications.

[0127] Administration and Dosage It is expected that the actual dose of β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) administered in accordance with the present invention may vary depending, for example, on the particular dosage form and method of administration. Many factors that may modulate the action of β-lactamase (e.g., body weight, sex, diet, time of administration, route of administration, excretion rate, medical condition of the subject, drug combinations, genetic disposition, and reaction sensitivities) can be considered by those of skill in the art. Administration can be continuous or in one or more separate doses up to the maximum tolerated dose. Optimal dosing rates for a given set of medical conditions can be ascertained by those of skill in the art using conventional dosage testing.

[0128] Individual doses of β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein, and variants thereof) may range, for example, from about 0.01 mg to about 1,000 mg, from about 0.01 mg to about 950 mg, from about 0.01 mg to about 900 mg, from about 0.01 mg to about 850 mg, from about 0.01 mg to about 800 mg, from about 0.01 mg to about 750 mg, from about 0.01 mg to about 700 mg, from about 0.01 mg to about 650 mg, from about 0.01 mg to about 600 mg, from about 0.01 mg to about 550 mg, from about 0.01 mg to about 500 mg, from about 0.01 mg to about 450 mg, from about 0.01 mg to about 400 mg, from about 0.01 mg to about 350 mg, It may be administered in a unit dosage form (e.g., tablet or capsule) containing about 0.01 mg to about 300 mg, about 0.01 mg to about 250 mg, about 0.01 mg to about 200 mg, about 0.01 mg to about 150 mg, about 0.01 mg to about 100 mg, about 0.1 mg to about 90 mg, about 0.1 mg to about 80 mg, about 0.1 mg to about 70 mg, about 0.1 mg to about 60 mg, about 0.1 mg to about 50 mg, about 0.1 mg to about 40 mg of active ingredient, about 0.1 mg to about 30 mg, about 0.1 mg to about 20 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 3 mg, about 0.1 mg to about 1 mg, or about 5 mg to about 80 mg per unit dosage form.For example, unit dosage forms may contain about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 55 mg, about 60 mg, about 75 mg, about 80 mg, about 95 mg, about 100 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 510 mg mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or about 1,000 mg (including all values ​​and ranges therebetween). In one embodiment, an individual dose of β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof) is administered in a unit dosage form containing 25 mg of β-lactamase. In another embodiment, an individual dose of β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof) is administered in a unit dosage form containing 50 mg of β-lactamase. In yet another embodiment, an individual dose of β-lactamase (e.g., SYN-004 or SYN-006, or other β-lactamase agents described herein and variants thereof) is administered in a unit dosage form containing 75 mg of β-lactamase.

[0129] In one embodiment, the β-lactamase is administered at a dose of about 0.01 mg to about 100 mg per day, about 0.01 mg to about 1,000 mg per day, about 0.01 mg to about 950 mg per day, about 0.01 mg to about 900 mg per day, about 0.01 mg to about 850 mg per day, about 0.01 mg to about 800 mg per day, about 0.01 mg to about 750 mg per day, about 0.01 mg to about 700 mg per day, about 0.01 mg to about 650 mg per day, or about 0.01 mg to about 100 mg per day. about 0.01mg to about 600mg per day, about 0.01mg to about 550mg per day, about 0.01mg to about 500mg per day, about 0.01mg to about 450mg per day, about 0.01mg to about 400mg per day, about 0.01mg to about 350mg per day, about 0.01mg to about 300mg per day, about 0.01mg to about 250mg per day, about 0.01mg to about 200mg per day, about 0.01mg to about 150mg per day, about 0.1 mg to about 100 mg, about 0.1 mg to about 95 mg per day, about 0.1 mg to about 90 mg per day, about 0.1 mg to about 85 mg per day, about 0.1 mg to about 80 mg per day, about 0.1 mg to about 75 mg per day, about 0.1 mg to about 70 mg per day, about 0.1 mg to about 65 mg per day, about 0.1 mg to about 60 mg per day, about 0.1 mg to about 55 mg per day, about 0.1 mg to about 50 mg per day, about 0.1 mg to about 45 mg per day mg, about 0.1 mg to about 40 mg per day, about 0.1 mg to about 35 mg per day, about 0.1 mg to about 30 mg per day, about 0.1 mg to about 25 mg per day, about 0.1 mg to about 20 mg per day, about 0.1 mg to about 15 mg per day, about 0.1 mg to about 10 mg per day, about 0.1 mg to about 5 mg per day, about 0.1 mg to about 3 mg per day, about 0.1 mg to about 1 mg per day, or about 5 mg to about 80 mg per day.

[0130] In various embodiments, the beta-lactamase is present in an amount of about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about and administered at a daily dose of about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or about 1,000 mg (including all values ​​and ranges therebetween).

[0131] In some embodiments, a suitable dose of a beta-lactamase (e.g., SYN-004 or SYN-006, or other beta-lactamase agents described herein, and variants thereof) is in the range of about 0.01 mg to about 100 mg per kg of the subject's body weight, e.g., 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, or about 100 mg per kg of body weight. mg, about 0.9 mg, about 1 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg (including all values ​​and ranges therebetween). In another embodiment, a suitable dose of beta-lactamase is in the range of about 0.01 mg / kg to about 10 mg / kg of body weight, about 0.01 mg / kg to about 9 mg / kg of body weight, about 0.01 mg / kg to about 8 mg / kg of body weight, about 0.01 mg / kg to about 7 mg / kg of body weight, about 0.01 mg / kg to about 6 mg / kg of body weight, about 0.05 mg / kg to about 5 mg / kg of body weight, about 0.05 mg / kg to about 4 mg / kg of body weight, about 0.05 mg / kg to about 3 mg / kg of body weight, about 0.05 mg / kg to about 2 mg / kg of body weight, about 0.05 mg / kg to about 1.5 mg / kg of body weight, or about 0.05 mg / kg to about 1 mg / kg of body weight.

[0132] In various embodiments, the dose of SYN-004 or SYN-006 is between about 75 mg and about 300 mg, e.g., about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, or about 300 mg.

[0133] According to some embodiments of the invention, the β-lactamase may be administered, for example, about once a day, about every two days, about every three days, about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every six months, or about once a year. In some embodiments, the β-lactamase may be administered more than once a day, for example, about twice, about three times, about four times, about five times, about six times, about seven times, about eight times, about nine times, or about ten times a day.

[0134] Additional Therapeutic Agents and Combination Therapies Administration of the microbiome protectant of the present invention may be combined with an additional therapeutic agent. Co-administration of the additional therapeutic agent and the present formulation may be simultaneous or sequential. Furthermore, the present formulation may include the additional therapeutic agent (e.g., co-formulated).

[0135] In some embodiments, the modified release formulations of the present invention are administered in combination with an additional therapeutic agent. In certain embodiments, the additional therapeutic agent and the microbiome protector may be combined in a single modified release formulation. In some embodiments, methods of treatment and / or prevention comprise administering a modified release formulation of the present invention to a subject undergoing treatment with an additional therapeutic agent.

[0136] In some embodiments, the additional therapeutic agent and the microbiome protector are administered to a subject simultaneously. As used herein, the term "simultaneously" means that the additional therapeutic agent and the microbiome protector are administered within a time interval of about 60 minutes, such as within about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute. The administration of the additional therapeutic agent and the microbiome protector can be by simultaneous administration of a single formulation (e.g., a formulation including the additional therapeutic agent and the microbiome protector) or separate formulations (e.g., a first formulation including the additional therapeutic agent and a second formulation including the microbiome protector).

[0137] Co-administration does not require that the additional therapeutic agents be administered simultaneously, as long as the timing of their administration is such that the pharmaceutical activities of the additional therapeutic agent and the microbiome protector overlap in time, thereby providing a combined therapeutic effect. For example, the additional agent and the microbiome protector can be administered sequentially. As used herein, the term "sequentially" means that the additional agent and the microbiome protector are administered at a time interval of at least about 60 minutes. For example, the sequential administration of the additional agent and the microbiome protector can be separated by at least about 60 minutes, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 1 day, at least about 2 days, at least about 3 days, or at least about 1 week. The optimal administration time may depend on the metabolic rate, excretion rate, and / or pharmacodynamic activity of the administered additional agent and microbiome protector. Either the additional agent or the microbiome protector can be administered first.

[0138] In other embodiments, the additional therapeutic agent and the microbiome protector are administered to a subject simultaneously, but release of the additional therapeutic agent and the microbiome protector from their respective dosage forms (or a single dosage form if co-formulated) in the GI tract occurs sequentially.

[0139] Co-administration also does not require that the additional therapeutic agents be administered to a subject by the same route of administration. Rather, each therapeutic agent can be administered by any suitable route, e.g., parenteral or non-parenteral.

[0140] In some embodiments, one or more microbiome protectants are used in methods to reduce the incidence and / or severity of complications associated with intravenous administration of β-lactam antibiotics to allo-HCT recipients, such as aGVHD and VRE colonization and / or VRE bloodstream infections.

[0141] In some embodiments, one or more microbiome protectants are used in combination with one or more therapeutic agents to prevent or treat one or more symptoms or side effects of diseases associated with allo-HCT, such as aGVHD and VRE colonization and / or VRE bloodstream infection. Such agents include, but are not limited to, immunosuppressants such as corticosteroids (methylprednisolone or prednisolone) and other immunosuppressive agents. An exemplary aGVHD treatment, in some embodiments, is prednisolone. Other exemplary aGVHD prevention and treatments include ibrutinib (e.g., IMBRUVICA), mycophenolate mofetil, mTOR inhibitors (such as sirolimus (rapamycin), everolimus), calcineurin inhibitors (such as tacrolimus or cyclosporine), cyclosporine, monoclonal antibodies (such as infliximab (e.g., REMICADE), tocilizumab (e.g., ACTEMRA), alemtuzumab (e.g., CAMPATH), basiliximab (e.g., SIMULECT), daclizumab (e.g., ZINBRYTA)), as well as denileukin diftitox (e.g., ONTAK), antithymocyte globulin (ATG), antilymphocyte globulin (ALG), pentostatin (e.g., NIPENT), ruxolitinib (e.g., JAKAFI), and photopheresis.

[0142] In some embodiments, the reduction in the incidence and / or severity of aGVHD by administration of β-lactamase inhibitors (e.g., ibrutinib (e.g., IMBRUVICA), mycophenolate mofetil, mTOR inhibitors (e.g., sirolimus (rapamycin), everolimus), calcineurin inhibitors (e.g., tacrolimus or cyclosporine), cyclosporine, monoclonal antibodies (e.g., infliximab (e.g., REMICADE), tocilizumab (e.g., ACTEMRA), alemtuzumab (e.g., CAMPATH), basiliximab This allows for a reduction in the dose or length or frequency of administration of additional treatments used to prevent or treat aGVHD, including methylprednisolone or prednisolone and other immunosuppressive agents, including methylprednisolone (e.g., SIMULECT), daclizumab (e.g., ZINBRYTA), etc., as well as denileukin diftitox (e.g., ONTAK), antithymocyte globulin (ATG), antilymphocyte globulin (ALG), pentostatin (e.g., NIPENT), ruxolitinib (e.g., JAKAFI), and photopheresis. For example, this may spare a patient from having to treat with high doses and frequent treatment of one or more steroids.

[0143] kit The present invention provides kits that can simplify the administration of the modified-release formulations described herein. The kits are collections of materials or components, including at least one of the modified-release formulations described herein. The exact nature of the components included in the kit depends on the intended purpose. In some embodiments, the kits are configured for the purpose of treating a human subject.

[0144] Instructions for use may be included in the kit. The instructions typically include specific language describing techniques to be used in using the components in the kit to affect a desired result, such as to treat a relevant disease described herein. Optionally, the kit also includes other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring devices, bandages, or other useful tools as would be readily recognized by one of ordinary skill in the art.

[0145] The materials and components assembled in the kit may be provided to the practitioner's storage location in any convenient and appropriate manner that preserves their operability and usefulness. For example, the components may be provided at room temperature, refrigerated temperature, or frozen temperature. The components are typically contained in suitable packaging material. In various embodiments, the packaging material is preferably constructed by well-known methods to provide a sterile, uncontaminated environment. The packaging material may have an exterior label indicating the contents and / or purpose of the kit and / or components.

[0146] definition As used herein, "a," "an," or "the" may mean one or more.

[0147] Additionally, the term "about" when used in connection with a reference numerical indication means the reference numerical indication plus or minus up to 10%. For example, the term "about 50%" covers a range of 45% to 55%.

[0148] An "effective amount," when used in connection with medical applications, is an amount effective to bring about a measurable treatment, prevention, or reduction in incidence of the desired disease.

[0149] As used herein, something is "reduced" if the activity and / or effect readout is reduced by a significant amount, e.g., about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 97% or more, about 98% or more, or more, including about 100%, in the presence of an agent or stimulus compared to the absence of such modulation. As will be understood by those of skill in the art, in some embodiments, activity will be reduced and some downstream readouts will be reduced, while others may increase.

[0150] Conversely, an activity is "increased" if the activity and / or effect readout is increased by a significant amount, e.g., by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 97% or more, about 98% or more, or less, including about 100% or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, about 50-fold or more, or about 100-fold or more, in the presence of an agent or stimulus compared to the absence of such agent or stimulus.

[0151] When referenced in this application, all component percentages are by weight of the total component unless otherwise specified. As used herein, the word "include" and variations thereof are intended to be non-limiting, and the recitation of items in a list does not exclude other similar items that may be useful in the compositions and methods of the present technology. Similarly, the words "can" and "may" and variations thereof are intended to be non-limiting, and a recitation that an embodiment can or may include particular elements or features does not exclude other embodiments of the present technology that do not include those elements or features.

[0152] The open-ended term "comprising," which is synonymous with terms such as include, containing, and having, is used herein to describe and claim the invention, present invention, or embodiments thereof, which may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

[0153] As used herein, the words "preferred" and "preferably" refer to embodiments of technology that provide certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the claimed technology.

[0154] The amounts of the components described herein required to achieve a therapeutic effect may be empirically determined according to conventional procedures for a particular purpose. Generally, for administration of a therapeutic agent (e.g., a β-lactamase described herein and / or an additional therapeutic agent) 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 an amount capable of achieving a desired result, particularly for the treatment of a disorder or disease. As used herein, an effective amount can include, for example, an amount sufficient to delay the onset of symptoms of a disorder or disease, alter the course of symptoms of a disorder or disease (e.g., slow the progression of symptoms of a disease), reduce or eliminate one or more symptoms or signs of a disorder or disease, or ameliorate symptoms of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of an underlying disease or disorder, regardless of whether an improvement is realized.

[0155] 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 approximately 50% of the population) and the ED50 (the dose therapeutically effective in approximately 50% of the population). Dosages can vary depending on the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. In some embodiments, compositions and methods with a large therapeutic index are preferred. The therapeutically effective dose is initially assessed by in vitro assays, including, for example, cell culture assays. A dose can also be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in a suitable animal model. Levels of the described compositions in plasma can be measured, for example, by high-performance liquid chromatography. The efficacy of any particular dosage can be monitored by a suitable bioassay. Dosages are determined by the physician and adjusted, if necessary, to accommodate the observed effects of treatment.

[0156] In certain embodiments, the effect will be a quantifiable change of about 10% or more, about 20% or more, about 30% or more, about 50% or more, about 70% or more, or about 90% or more. In some embodiments, the effect will be a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or about 90% or more. Therapeutic benefit also includes halting or slowing the progression of an underlying disease or disorder, regardless of whether an improvement is perceived.

[0157] As used herein, "method of treatment" applies equally to the use of a composition for the treatment of a disease or disorder described herein, and / or the composition for use, and / or the use in the manufacture of a medicament for the treatment of a disease or disorder described herein. [Example]

[0158] Example 1: SYN-004 (P3A) reduced and / or decreased piperacillin levels in mouse feces The purpose of this experiment was to determine whether a liquid formulation of SYN-004 (e.g., β-lactamase in phosphate-buffered saline) could be delivered to the small and large intestine of mice by oral gavage.

[0159] Mice received 500 mg / kg piperacillin / tazobactam subcutaneously twice daily for two days. The mice were then divided into two cohorts: (1) a first cohort received 500 mg / kg piperacillin / tazobactam subcutaneously twice daily for two days but did not receive oral gavage of a liquid formulation of P3A (e.g., β-lactamase in saline); and (2) a second cohort received 500 mg / kg piperacillin / tazobactam subcutaneously twice daily for two days and oral gavage of 10 mg / kg P3A twice daily for two days. The control cohort of mice received neither piperacillin / tazobactam nor oral gavage of P3A.

[0160] After two days, the feces of the mice were evaluated for the presence of piperacillin. Figure 1 shows that the amount of piperacillin (µg per gram of feces) decreased in mice receiving 500 mg / kg piperacillin / tazobactam subcutaneously twice daily for two days and a liquid formulation of P3A (e.g., β-lactamase in phosphate-buffered saline) administered orally twice daily for two days. The leftmost bar in the bar graph corresponds to data from a cohort of control mice, the middle bar corresponds to data from a first cohort of mice receiving piperacillin / tazobactam but not P3A, and the rightmost bar corresponds to data from a second cohort of mice receiving both piperacillin / tazobactam and P3A.

[0161] Example 2: SYN-004 (P3A) protected the Firmicutes bacterial phylum from piperacillin / tazobactam Mice received 125 mg / kg piperacillin / tazobactam subcutaneously twice daily for two days. The mice were then divided into two cohorts: (1) a first cohort received 125 mg / kg piperacillin / tazobactam subcutaneously twice daily for two days but did not receive oral gavage of a liquid formulation of P3A (e.g., β-lactamase in saline); and (2) a second cohort received 125 mg / kg piperacillin / tazobactam subcutaneously twice daily for two days and oral gavage of 10 mg / kg P3A twice daily for two days. Mice in the control cohort received neither piperacillin / tazobactam nor oral gavage of P3A. A separate control cohort of mice received P3A alone.

[0162] Two days later, 16S sequencing of mouse feces was performed to assess the levels of Firmicutes (e.g., G+ anaerobes, including Lactobacillus and Clostridium, which produce short-chain fatty acids) in the feces. Figure 2 shows that piperacillin / tazobactam alone reduced the abundance of Firmicutes in mouse feces, while the presence of P3A restored and protected the fold abundance of Firmicutes. The leftmost bar in the bar graph corresponds to data from a cohort of control mice that received neither drug nor β-lactamase, the second bar from the left corresponds to data from a cohort of mice that received P3A only, the second bar from the right corresponds to data from a first cohort of mice that received piperacillin / tazobactam but not a liquid formulation of P3A (e.g., β-lactamase in phosphate-buffered saline solution), and the rightmost bar in the bar graph corresponds to data from a second cohort of mice that received both piperacillin / tazobactam and P3A.

[0163] 16S sequence analysis was also performed to assess the total bacterial copy number present per gram of mouse feces. Figure 3 shows that piperacillin / tazobactam reduced the total bacterial copy number present in mouse feces, and this reduction was mitigated when piperacillin / tazobactam was administered together with P3A. The leftmost bar in the bar graph corresponds to data from a cohort of control mice that received neither drug nor β-lactamase; the second bar from the left corresponds to data from a cohort of mice that received P3A alone; the second bar from the right corresponds to data from a first cohort of mice that received piperacillin / tazobactam but not a liquid formulation of P3A (e.g., β-lactamase in phosphate-buffered saline solution); and the rightmost bar corresponds to data from a second cohort of mice that received both piperacillin / tazobactam and P3A.

[0164] Example 3: SYN-004 (P3A) reduced antibiotic-exacerbated GVHD in a model organism This study was conducted to evaluate the effect of SYN-004 administration on protecting against and / or reducing the severity of GVHD associated with bone marrow transplantation.

[0165] 129S1 mice were lethally irradiated and then transplanted with bone marrow cells (B6 donor bone marrow cells) at 5 months and with a defined dose of splenic T lymphocytes from C57BL / 6 mice at 2 months. Both mice were MHC type H2 b Since they share the same MHC, they represent a clinically relevant MHC-matched transplant model.

[0166] Mice were divided into four cohorts: (1) a cohort that received only B6 donor bone marrow cells at 5 months; (2) a cohort that received B6 donor bone marrow cells at 5 months, T cell transplants at 2 months, and 100 mg / kg piperacillin / tazobactam subcutaneously three times a week starting 10 days after bone marrow transplantation, and 20 mg / kg of a liquid formulation of P3A (e.g., β-lactamase in phosphate-buffered saline) administered by oral gavage three times a week starting 10 days after bone marrow transplantation simultaneously with the piperacillin / tazobactam administration; (3) a cohort that received B6 donor bone marrow cells and T cell transplants; and (4) a cohort that received B6 donor bone marrow cells at 5 months, T cell transplants at 2 months, and 100 mg / kg of piperacillin / tazobactam subcutaneously three times a week starting 10 days after bone marrow transplantation.

[0167] Figure 4 shows the results of a survival study with an endpoint of 30 days after bone marrow transplantation. The data demonstrate that P3A administration reduced antibiotic-exacerbated GVHD associated with bone marrow transplantation in terms of reduced mortality.

[0168] Example 4: SYN-004 (P3A) prevented the loss of gut microbiota diversity and intestinal dysbiosis in a model organism This experiment was conducted to evaluate the effect of SYN-004 administration on modulating and / or reducing Enterococcus monodominance. This experiment was also designed to evaluate whether SYN-004 administration could prevent the decline in microbiome diversity in lethally irradiated mice undergoing bone marrow transplantation.

[0169] 129S1 mice were lethally irradiated and then transplanted with bone marrow cells (B6 donor bone marrow cells) at 5 months and with a defined dose of splenic T lymphocytes from C57BL / 6 mice at 2 months. Mice were divided into three cohorts: (1) a cohort receiving saline, (2) a cohort receiving piperacillin / tazobactam, and (3) a cohort receiving piperacillin / tazobactam (Zosyn, abbreviated ZO) and SYN-004 (P3A; abbreviated BlacT).

[0170] Fecal samples were collected 21 days after transplantation and subjected to 16S sequencing. Figures 5 and 6 show the results of the 16S sequencing analysis, which demonstrate that fecal samples from mice administered both piperacillin / tazobactam and SYN-004 showed reduced monodominance of Enterococcus faecalis compared to fecal samples from mice administered piperacillin / tazobactam alone. In Figure 6, empty bars indicate that a particular sample was not amplified.

[0171] Example 5: SYN-006 (P2A) protected the gut microbiome of a model organism from β-lactam antibiotics This experiment was conducted to determine whether a liquid formulation of SYN-006 (e.g., β-lactamase in phosphate-buffered saline) could be delivered to the small and large intestines of mice by oral gavage.

[0172] Mice received 100 mg / kg imipenem plus cilastatin subcutaneously for 2 days, and fecal samples were collected on day 3. Mice were simultaneously divided into (1) a first cohort that received 100 mg / kg imipenem plus cilastatin subcutaneously for 2 days but did not receive oral gavage of a liquid formulation of P2A (e.g., β-lactamase in saline); (2) a second cohort that received 100 mg / kg imipenem plus cilastatin subcutaneously for 2 days and oral gavage of 1 mg / kg P2A twice daily for 2 days; (3) a third cohort received 100 mg / kg imipenem plus cilastatin subcutaneously for two days and 10 mg / kg P2A by oral gavage twice daily for two days; and (4) a fourth cohort received 100 mg / kg imipenem plus cilastatin subcutaneously for two days and 50 mg / kg P2A by oral gavage twice daily for two days.

[0173] Mouse feces were collected on day 3. 16S sequencing of mouse feces was performed to assess the levels of Firmicutes bacteria (e.g., G+ anaerobes, including Lactobacillus and Clostridium, which produce short-chain fatty acids) in mouse feces. Figure 7 shows that the fold abundance of Firmicutes was restored and protected in the presence of both imipenem and cilastatin and P2A at 10 mg / kg and 50 mg / kg doses compared to the presence of imipenem and cilastatin alone. The leftmost bar of the bar graph corresponds to data from the cohort of mice that received imipenem + cilastatin only, the second bar from the left corresponds to data from the second cohort of mice that received imipenem + cilastatin and 1 mg / kg P2A, the third bar from the left corresponds to data from the third cohort of mice that received imipenem + cilastatin and 10 mg / kg P2A, and the rightmost bar of the bar graph corresponds to data from the fourth cohort of mice that received imipenem + cilastatin and 50 mg / kg P2A.

[0174] Next, experiments were conducted to evaluate the effect of SYN-006(P2A) administration on modulating and / or reducing the overgrowth of Enterococcus monodominance, particularly Enterococcus faecium, in imipenem-treated mice.

[0175] Mice received 100 mg / kg imipenem plus cilastatin subcutaneously for 2 days, and fecal samples were collected on day 3. Mice were simultaneously divided into (1) a first cohort that received 100 mg / kg imipenem plus cilastatin subcutaneously for 2 days but did not receive oral gavage of a liquid formulation of P2A (e.g., β-lactamase in saline); (2) a second cohort that received 100 mg / kg imipenem plus cilastatin subcutaneously for 2 days and oral gavage of 1 mg / kg P2A twice daily for 2 days; The mice were divided into four cohorts: (3) a third cohort received 100 mg / kg imipenem plus cilastatin subcutaneously for two days and 10 mg / kg P2A twice daily by oral gavage for two days; and (4) a fourth cohort received 100 mg / kg imipenem plus cilastatin subcutaneously for two days and 50 mg / kg P2A twice daily by oral gavage for two days. Fecal samples were collected on day 3 and subjected to 16S sequencing. Figure 8 shows the results of 16S sequencing, indicating that fecal samples from mice receiving both imipenem plus cilastatin and SYN-006 (10 mg / kg and 50 mg / kg doses) showed reduced monodominance of Enterococcus faecalis compared to fecal samples from mice receiving imipenem plus cilastatin alone. Figure 9 shows the relative abundance of Enterococcus faecium in the feces of mice treated with imipenem plus cilastatin alone or with P2A at doses of 1 mg / kg, 10 mg / kg, and 50 mg / kg. The results show that SYN-006 (P2A) prevents the overgrowth of Enterococcus faecium in imipenem-treated mice. In each histogram, the cluster on the left represents pre-treatment values, and the cluster on the right represents post-treatment values.

[0176] Example 6: SYN-006 (P2A) reduced antibiotic-exacerbated GVHD in a model organism This experiment was conducted to evaluate the effect of SYN-006 (P2A) administration on protecting against and / or reducing the severity of GVHD associated with bone marrow transplantation.

[0177] 129S1 mice were lethally irradiated and then transplanted with bone marrow cells (B6 donor bone marrow cells) at 5 months and with a defined dose of splenic T lymphocytes from C57BL / 6 mice at 2 months. Both mice were MHC type H2 b Since they share the same MHC, they represent a clinically relevant MHC-matched transplant model.

[0178] Mice were divided into three cohorts: (1) a cohort that received only B6 donor bone marrow cells for 5 months and T cells for 2 months; (2) a cohort that received B6 donor bone marrow cells for 5 months, T cells for 2 months, and 100 mg / kg imipenem plus cilastatin subcutaneously three times per week starting 10-25 days after bone marrow transplant; and (3) a cohort that received B6 donor bone marrow cells for 5 months, T cells for 2 months, 100 mg / kg imipenem plus cilastatin subcutaneously three times per week starting 10-25 days after bone marrow transplant, and 50 mg / kg P2A liquid formulation (e.g., β-lactamase in phosphate-buffered saline) by oral gavage three times per week starting 10-25 days after bone marrow transplant.

[0179] Figure 10 shows the results of a survival study with an endpoint of 40 days after bone marrow transplantation. The data indicate that antibiotic-exacerbated GVHD associated with bone marrow transplantation was reduced by P2A administration in terms of reduced mortality.

[0180] While the invention has been described in relation to specific embodiments thereof, it will be understood that further modifications are possible, and this application is generally intended to cover any variation, use, or modification of the invention in accordance with the principles of the invention, including such departures from the present disclosure as are within known or customary practice within the art to which the invention pertains, and which may apply the essential features set forth above and in the following appended claims.

[0181] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed by the following claims.

[0182] Incorporation by Reference All patents and publications mentioned herein are incorporated by reference in their entirety. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is entitled to antedate such publication by virtue of prior invention.

[0183] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of an individual section is equally applicable to all sections.

Claims

1. A method of reducing the incidence and / or severity of graft-versus-host disease (GVHD) in a subject in need thereof, comprising administering an effective amount of a beta-lactamase agent.

2. 2. The method of claim 1, wherein the β-lactamase agent has an amino acid sequence having 95% or greater identity to SEQ ID NO:1 or SEQ ID NO:

6.

3. 2. The method of claim 1, wherein the β-lactamase agent has the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:

6.

4. The method of claim 1 , wherein the subject is a transplant recipient.

5. The method of claim 1, wherein the subject is a recipient of an allogeneic hematopoietic stem cell transplant.

6. The method according to any one of claims 1 to 5, wherein the subject is a recipient of bone marrow cells, peripheral blood cells, or umbilical cord cells.

7. The method according to any one of claims 1 to 6, wherein the subject is receiving or has been receiving intravenously administered a β-lactam antibiotic.

8. 8. The method of claim 7, wherein the β-lactam antibiotic is selected from penicillins (e.g., piperacillin / tazobactam), cephalosporins (e.g., cefepime), and / or carbapenems (e.g., meropenem, imipenem / cilastatin).

9. The method according to any one of claims 1 to 8, wherein the GVHD is acute.

10. The method of any of claims 4 to 9, wherein the beta-lactamase is administered prior to transplantation.

11. The method of any of claims 4 to 10, wherein the beta-lactamase is administered subsequent to transplantation.

12. 12. The method of any one of claims 1 to 11, wherein intestinal dysbiosis is reduced or eliminated in the subject.

13. 13. The method of any one of claims 1 to 12, wherein new colonization within the subject's intestinal microbiome is prevented.

14. 14. The method of any one of claims 1 to 13, wherein the spread of colonization within the gut microbiome of the subject is prevented.

15. 15. The method according to any one of claims 1 to 14, wherein monodominance of the intestinal microbiome is prevented.

16. 16. The method of any one of claims 13 to 15, wherein the colonization, expansion of colonization, or monodominance of the microbiome comprises one or more multidrug-resistant organisms.

17. The one or more multidrug-resistant organisms may be selected from the group consisting of Aeromonas hydrophila, Bacillus (e.g., Bacillus cereus), Bifidobacterium, Bordetella, Borrelia, Brucella, Burkholderia, C. difficile, Campylobacter (e.g., Campylobacter fetus and Campylobacter jejuni), Chlamydia, Chlamydophila, Clostridium (e.g., Clostridium botulinum), and the like. botulinum), Clostridioides difficile (formerly Clostridium difficile), and Clostridium perfringensperfringens), Corynebacterium, Coxiella, Ehrlichia, Enterobacteriaceae (e.g., carbapenem-resistant Enterobacteriaceae (CRE) and extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E)), fluoroquinolone-resistant Enterobacteriaceae, Enterococcus (e.g., vancomycin-resistant Enterococcus spp., extended-spectrum β-lactam-resistant Enterococci (ESBL-E)), , and vancomycin-resistant enterococci (VRE)), Escherichia (e.g., enteroaggregative E. coli, enterohemorrhagic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterotoxigenic E. coli (e.g., but not limited to, LT and / or ST), E. coli 0157:1-17, and multidrug-resistant E. coli), Francisella, Haemophilus, Helicobacter (e.g., Helicobacter pylori), pylori), Klebsiella (e.g., Klebsiellia pneumonia and multidrug-resistant Klebsiella), Legionella, Leptospira, Listeria (e.g., Lysteria monocytogenes), Morganella, Mycobacterium, Mycoplasma, Neisseria, Orientia, Plesiomonas shigelloides, antibiotic-resistant Proteobacteria, Proteus, Pseudomonas, Rickettsia, Salmonella (e.g., Salmonella paratyphi), paratyphi, Salmonella species, and Salmonella typhityphi), Shigella (e.g., Shigella spp.), Staphylococcus (e.g., Staphylococcus aureus and Staphylococcus spp.), Streptococcus, Treponema, Vibrio (e.g., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio spp., and Vibrio vulnificus), and Yersinia (e.g., Yersinia enterocolitica).

18. 18. The method of claim 17, wherein the microbiome colonization, expansion of colonization, or monodominance comprises an overgrowth of Enterococcus species.

19. 20. The method of claim 18, wherein the microbiome colonization, expansion of colonization, or monodominance comprises vancomycin-resistant enterococci (VRE).

20. The method of any of claims 1 to 19, wherein the dose or length or frequency of administration of a therapeutic agent for the treatment of GVHD is reduced.

21. 21. The method of claim 20, wherein the therapeutic agent for the treatment of GVHD is a steroid.

22. 22. The method of claim 21, wherein the therapeutic agent for the treatment of GVHD is methylprednisolone or prednisone.

23. 23. The method of any one of claims 1 to 22, wherein the beta-lactamase agent is released in the small intestine.

24. 23. The method of claim 22, wherein the beta-lactamase agent is released in one or more of the duodenum, jejunum, ileum, and / or ileocecal junction.

25. 22. The method of any one of claims 1 to 21, wherein the beta-lactamase agent is released in the large intestine.

26. 25. The method of claim 24, wherein the beta-lactamase agent is released in at least one or more of the cecum, the ascending, transverse, descending, or sigmoid portion of the colon, and the rectum.

27. 27. The method of any one of claims 1-26, wherein the β-lactamase agent is formulated with a modified release coating that is substantially stable in gastric fluids.

28. 28. The method of any one of claims 1-27, wherein the β-lactamase agent is formulated with a modified release coating that has pH-dependent solubility.

29. 29. The method of any one of claims 1-28, wherein the β-lactamase agent is formulated with a modified release coating having a time-dependent erosion profile.

30. 30. The method of any one of claims 1 to 29, wherein the β-lactamase agent is formulated with a modified release coating that is degraded by enzymes of microorganisms present in intestinal flora.

31. 31. The method of any one of claims 1 to 30, wherein the beta-lactamase agent is formulated as a capsule or tablet.

32. 32. The method of any one of claims 1 to 31, wherein the beta-lactamase agent is formulated for oral administration.

33. A method for preventing or reducing the incidence of colonization, spread of colonization, or monodominance, and / or infection by one or more multidrug-resistant pathogens in a subject who is a transplant recipient, comprising administering an effective amount of a beta-lactamase agent.

34. 34. The method of claim 33, wherein the beta-lactamase agent has an amino acid sequence having 95% or greater identity to SEQ ID NO:1 or SEQ ID NO:

6.

35. 34. The method of claim 33, wherein the beta-lactamase agent has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

6.

36. 34. The method of claim 33, wherein the subject has experienced a prolonged hospital stay and / or is at increased risk of mortality from the colonization and / or infection.

37. 34. The method of claim 33, wherein the subject is a recipient of an allogeneic hematopoietic stem cell transplant.

38. The method of any one of claims 33 to 37, wherein the subject is a recipient of bone marrow cells, peripheral blood cells, or umbilical cord cells.

39. The method of any one of claims 33 to 38, wherein the subject is receiving or has been receiving intravenously a β-lactam antibiotic.

40. 40. The method of claim 39, wherein the beta-lactam antibiotic is selected from penicillins (piperacillin / tazobactam), cephalosporins (cefepime), imipenem / cilastatin, and meropenem.

41. The method of any of claims 33 to 40, wherein the beta-lactamase is administered prior to transplantation.

42. 42. The method of any of claims 36-41, wherein the beta-lactamase is administered subsequent to transplantation.

43. 43. The method of any one of claims 33 to 42, wherein intestinal dysbiosis is reduced or eliminated in the subject.

44. 44. The method of any one of claims 33 to 43, wherein new colonization within the subject's intestinal microbiome is prevented.

45. 45. The method of any one of claims 33 to 44, wherein the spread of colonization within the gut microbiome of the subject is prevented.

46. 46. ​​The method of any one of claims 33 to 45, wherein monodominance of the intestinal microbiome is prevented.

47. 47. The method of any one of claims 33 to 46, wherein the microbiome colonization, expansion of colonization, or monodominance comprises one or more multidrug resistant organisms.

48. The one or more multidrug resistant organisms may be selected from the group consisting of Aeromonas hydrophila, Bacillus (e.g., Bacillus cereus), Bifidobacterium, Bordetella, Borrelia, Brucella, Burkholderia, C. difficile, Campylobacter (e.g., Campylobacter fetus and Campylobacter jejuni), Chlamydia, Chlamydophila, Clostridium (e.g., Clostridium botulinum), and the like. botulinum), Clostridioides difficile (formerly Clostridium difficile), and Clostridium perfringensperfringens), Corynebacterium, Coxiella, Ehrlichia, Enterobacteriaceae (e.g., carbapenem-resistant Enterobacteriaceae (CRE) and extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E)), fluoroquinolone-resistant Enterobacteriaceae, Enterococcus (e.g., vancomycin-resistant Enterococcus spp., extended-spectrum β-lactam-resistant Enterococci (ESBL-E)), , and vancomycin-resistant enterococci (VRE)), Escherichia (e.g., enteroaggregative E. coli, enterohemorrhagic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterotoxigenic E. coli (e.g., but not limited to, LT and / or ST), E. coli 0157:1-17, and multidrug-resistant E. coli), Francisella, Haemophilus, Helicobacter (e.g., Helicobacter pylori), pylori), Klebsiella (e.g., Klebsiellia pneumonia and multidrug-resistant Klebsiella), Legionella, Leptospira, Listeria (e.g., Lysteria monocytogenes), Morganella, Mycobacterium, Mycoplasma, Neisseria, Orientia, Plesiomonas shigelloides, antibiotic-resistant Proteobacteria, Proteus, Pseudomonas, Rickettsia, Salmonella (e.g., Salmonella paratyphi), paratyphi, Salmonella species, and Salmonella typhityphi), Shigella (e.g., Shigella spp.), Staphylococcus (e.g., Staphylococcus aureus and Staphylococcus spp.), Streptococcus, Treponema, Vibrio (e.g., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio spp., and Vibrio vulnificus), and Yersinia (e.g., Yersinia enterocolitica).

49. 49. The method of any one of claims 33 to 48, wherein intestinal colonization by VRE is reduced.

50. 50. The method of any one of claims 33 to 49, wherein bloodstream infections caused by VRE are reduced.

51. The method according to any one of claims 33 to 50, wherein the dose or the length or frequency of administration of a therapeutic agent for the treatment of VRE colonization and / or infection is reduced.

52. 49. The method of any one of claims 33 to 48, wherein intestinal colonization by Carbapenem-Resistant Enterobacteriaceae (CRE) is reduced.

53. 50. The method of any one of claims 33 to 49, wherein CRE bloodstream infections are reduced.

54. The method according to any one of claims 52 to 53, wherein the dose or the length or frequency of administration of a therapeutic agent for the treatment of CRE colonization and / or infection is reduced.

55. 55. The method of any one of claims 1-54, wherein the beta-lactamase agent is released in the small intestine.

56. 56. The method of claim 55, wherein the beta-lactamase agent is released in one or more of the duodenum, jejunum, ileum, and / or ileocecal junction.

57. 55. The method of any of claims 33-54, wherein the beta-lactamase agent is released in the large intestine.

58. 58. The method of claim 57, wherein the beta-lactamase agent is released in one or more of the cecum, the ascending, transverse, or sigmoid portion of the colon, and the rectum.

59. 59. The method of any one of claims 1-58, wherein the β-lactamase agent is formulated with a modified release coating that is substantially stable in gastric fluids.

60. 60. The method of any one of claims 1-59, wherein the β-lactamase agent is formulated with a modified release coating that has pH-dependent solubility.

61. 61. The method of any one of claims 1-60, wherein the β-lactamase agent is formulated with a modified release coating having a time-dependent erosion profile.

62. 62. The method of any one of claims 1 to 61, wherein the β-lactamase agent is formulated with a modified release coating that is degraded by enzymes of microorganisms present in the intestinal flora.

63. 63. The method of any one of claims 1-62, wherein the beta-lactamase agent is formulated as a capsule or tablet.

64. 64. The method of any one of claims 1-63, wherein the beta-lactamase agent is formulated for oral administration.

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