Compositions and methods for phage-based treatment of vibrio infections

Abstract

Formulations of lytic bacteriophages against Vibrio cells and use thereof for preventing or treating bacterial infections, including those by bacteria of genus Vibrio, for example, V. cholerae. The bacteriophage compositions contain three strains of lytic bacteriophage against Vibrio cells. The compositions include Vibrio bacteriophages ICP1, ICP2, and ICP3; and an excipient coating disposed on the bacteriophages, the coating including hydroxypropyl β-cyclodextrin, HPC, HPMC, HPMC-AS, or PVP-VA.

Description

COMPOSITIONS AND METHODS FOR PHAGE-BASED TREATMENT OF VIBRIO INFECTIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application 63 / 677,643, filed July 31, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with U.S. government support under grant R44AI142960 awarded by the National Institutes of Health. The federal government has certain rights in the invention.TECHNICAL FIELD

[0003] The invention relates to phage compositions useful for treatment of cholera.BACKGROUND

[0004] Cholera is a diarrheal disease caused by multiplication and toxin production by Vibrio cholerae in the small intestine. Cholera remains a substantial global health burden and is endemic to many parts of Africa and Asia (Zuckerman et al. The Lancet Infectious Diseases 7, 521-530, 2007). Recent widespread epidemics in disaster-stricken or war-torn countries such as Haiti (Luquero et al. Emerging infectious diseases 22, 410-416, 2016) and Iraq (Bagcchi, S. The Lancet Infectious Diseases 16, 24-25, 2016) highlight the vulnerabilities of populations to sudden outbreaks. Current recommended preventatives include mass vaccinations with the WHO-prequalified oral cholera vaccine (Qadri et al. The New England J Med 374, 1723-1732, 2016) and increased awareness of sanitation and hygiene practices (Taylor et al. PloS One 10, e0135676, 2015). Access to clean water, however, is difficult, and vaccination campaigns require forethought and time for efficacy; both methods are notlogistica lly feasible for immediate protection in the event of an outbreak. Prophylactic antibiotic use is discouraged because of disruptions to the normal microbiota, increasing of antibiotics resistance, and other agents not being readily available to treat or prevent this disease.

[0005] Three strains of vegetative (lytic or virulent) Vibrio bacteriophages have been isolated and sequenced, ICP1, ICP2, and ICP3. (Seed et al., mBio, 2(l):l-9, 2011).

[0006] A few strains of V. cholerae have a resistance mechanism against ICP1 (Seed et aL, Nature, 494 (7438): 489-491, 2013). However, ICP1 phage strains have evolved a mechanism to overcome that resistance by a CRISPR / Cas system which allows the phage to maintain an infectious cycle.

[0007] The bacterial receptor for ICP2 is a surface protein critical for virulence called OmpU (Seed et al., eLife, 3:e03497, 2014). Bacterial escape mutant strains were observed in a portion of patients and contain point mutations in the gene that encodes OmpU protein. Certain patients were observed to shed avirulent V. cholerae escape mutants that no longer express the OmpU protein, these mutants not transmitting disease to other people.

[0008] It was found that using a plurality of bacteriophage strains avoids proliferation of resistant bacteria strains. See US 10,953,053. Specifically, the virions of the bacteriophage bind the surface of the Vibrio cell by attachment of the first strain to a first receptor on the bacterial surface, and attachment of the second strain to a second receptor, and the first and second receptors are molecularly different. In this way, the rate of spontaneous mutation to resistance, which is on the order of a frequency in a bacterial population of about 10“7to about 10“9for most bacterial genes, would need to be squared to obtain a double resistance mutant to two phage strains, which is a rate so low as to be negligible or even non-existent. Accordingly in embodiments of the composition herein, the virions of the first strain of bacteriophage bind to bacterial cell outer membrane protein OmpU andthe virions of the second strain of bacteriophage bind to lipopolysaccharide. Also, cells of strains of a normal microbiome lack receptors for the plurality of the bacteriophage strains, and the viabilities of the normal gut flora species remain unaffected.

[0009] Previously, the ICP bacteriophage mixtures were diluted in 2.5% sodium bicarbonate and orogastrically dosed by oral intubation. US 10,953,053. Administering bacteriophages in liquid form is not the most effective means of treatment, with clinical trials yielding mixed results. Problems associated with liquid formulations include difficulty in applying a liquid preparation to a site of infection, adverse conditions brought about by the body's natural physio-chemical environment, and the subject's immune response. Rosner and Clark, Pharmaceuticals 2021, 14, 359. https: / / doi.org / 10.3390 / phl4040359.

[0010] While various solid formulations of bacteriophages have been developed, there remains a need for production processes and resulting bacteriophage formulations that address the problems of bacteriophages protein mis-folding and aggregation and denaturization, sensitivity of phages to organic solvents, pH, temperature, and salinity, highly variable survivability across different bacteriophages, limited storage stability at room temperature or without freezing. Notably, bacteriophage formulations may be sensitive to the highly acidic environment of the stomach, and thermal stresses. Formulation production process may also result in bacteriophage degradation.SUMMARY

[0011] The invention includes a composition for preventing or reducing a Vibrio bacterial infection in a subject, the composition containing Vibrio bacteriophages ICP1, ICP2, and ICP3; and an excipient coating disposed on the bacteriophages. The excipient coating comprises hydroxypropyl p-cyclodextrin, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose-acetate succinate (HPMC-AS), or polyvinylpyrrolidone-vinyl acetate (PVP-VA).

[0012] In an advantageous aspect, the Vibrio bacteriophages are present in substantially equal amounts.

[0013] In yet another aspect, the excipient coating includes a sugar or amino acid. The sugar may be, for example, trehalose, sucrose, or mannitol. The amino acid may be, for example, leucine.

[0014] In another aspect, the excipient coating comprises hydroxypropyl 0-cyclodextrin or HPC. In yet another aspect, the excipient coating comprises hydroxypropyl 0-cyclodextrin and trehalose.

[0015] In another aspect, the sugar is absent, or is present in a ratio of from 0:100 to 100:0 or 10:90 to 90:10 sugar: hydroxypropyl |3-cyclodextrin, HPC, HPMC, HPMC-AS, or PVP- VA. In yet another aspect, the sugar is present in a ratio of from 45:55 to 55:45 sugar: hydroxypropyl [J-cyclodextrin, HPC, HPMC, HPMC-AS, or PVP-VA.

[0016] In another aspect, the composition is made by a spray-drying or fluidized bed coating process.

[0017] In another aspect, the invention is a method of preventing or treating a bacterial infection in a subject by administering an effective amount of the compositions as described above.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows the results of stability testing of a formulation of ICP1 and 50:50 trehalose and hydroxypropyl P-cyclodextrin.

[0019] FIG. 2 shows the results of stability testing of a formulation of ICP1 and 50:50 sucrose and HPC.

[0020] FIG. 3 shows the results of stability testing of a formulation of ICP2 and 50:50 trehalose and hydroxypropyl |3-cyclodextrin.

[0021] FIG. 4 shows the results of stability testing of a formulation of ICP1 and 50:50 sucrose and HPC.

[0022] FIG. 5 shows the results of stability testing of a formulation of ICP3 and trehalose.

[0023] FIG. 6 shows the results of stability testing of a formulation of ICP3 and 50:50 sucrose / HPC.

[0024] FIG. 7 shows the results of testing different phage ratios in infant mice.

[0025] FIG. 8 shows the results of efficacy testing for V. cholerae removal in infant mice of phage mixtures in spray-dried PVC versus non-processed PVC.DETAILED DESCRIPTION

[0026] Those skilled in the art will understand that this invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth in this application. Rather, these embodiments are provided so that this disclosure will fully convey the invention to those skilled in the art. Many modifications and other embodiments of the invention will come to mind in one skilled in the art to which this invention pertains having the benefit of the teachings presented herein.

[0027] The word "phage" as used herein is a shortened form of and is synonymous with the term "bacteriophage" referring to bacterial-specific viruses.

[0028] A "pharmaceutically acceptable carrier" includes any additive, adjuvant, diluent, or excipient used for pharmaceutical or nutraceutical dosage forms.

[0029] Formulations for oral administration include capsules, tablets, pills, powders, and granules. Such solid dosage forms might include a pharmaceutically acceptable excipient or carrier, e.g., fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; humectants such as glycerol; disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents such as cetyl alcohol and glycerol monostearate; absorbents such as kaolin and bentonite clay; and lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Capsules, tablets, and pills may also contain buffering agents.

[0030] The excipients disclosed herein as coating the Vibrio bacteriophage compositions have not been previously disclosed for use in solid compositions as described herein. Hydroxypropyl cellulose (HPC) and hydroxypropyl methylcellulose (HPMC) have been tried with hydrogel formulations as phage delivery vehicle (see doi.Org / 10.1016 / j.ijpharm.2021.120850) but not for use in solid compositions, much less those made by spray-drying or fluidized bed coating (FBC). Previous disclosures have not been found of hydroxypropyl |3-cyclodextrin, HPMC-AS, and PVP-VA used in phage compositions as described herein.

[0031] Soft and hard-filled gelatin capsules may include excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols. Tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art.

[0032] In one aspect, the pharmaceutically acceptable composition may be administered with or without food.

[0033] The term "treating" or "treatment" means reversing, alleviating, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof. In some aspects, treatment may be administered after one or more symptoms have developed. In other aspects, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., due to a history of symptoms and / or due to genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. "Preventing" or "prevention" refers to use as a prophylactic for reducing the risk of acquiring a disease or disorder, i.e ., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease. The subject of treatment is a mammal, preferably a human subject.

[0034] Vibrio bacteriophages ICP1, ICP2, and ICP3 may be obtained by methods known in the field. The terms "ICP1" and "ICP2" and "ICP3" include variants containing nucleotide mutations of up to 5%, or preferably 4%, 3%, 2%, 1%, or less than 1%, in particular where anti-bacterial activity is not significantly affected.

[0035] The Vibrio bacteriophages ICP1, ICP2, and ICP3 may be present in the compositions described herein in substantially equal amounts. By "substantially equal amounts" is meant that each phage represents 30% to 36%, preferably 33% to 34% of the total amount of Vibrio phage in the composition, measured by mass or another standard method in the field. In certain embodiments, the relative amounts of each phage may vary significantly. For example, each phage may represent 8% to 85%, or any relative amounts between substantially equal amounts of each phage and 8% to 85% of each phage, preferably 15% to 59% of the total amount of phage in the composition.

[0036] The compositions described herein may further contain an enteric coating material disposed on the excipient coating. The enteric coating material may be any of the various enteric coating excipients used for solid pharmaceutical formulations for oral administration.

[0037] The composition advantageously comprises solid particles made by spray-drying an aqueous suspension of the Vibrio bacteriophages and excipients. The composition may also be made by a fluidized bed coating (FBC) process, by atomizing a solution of the Vibrio bacteriophages into a Wurster column, then introducing the excipients into the Wurster column, followed by Wurster coating the bacteriophages with the excipient coating.

[0038] The beads used in this FBC process are made of sugar and starch, and are referred to interchangeably in the field as, e.g., "sugar spheres" or "sugar pellets" or "sugar beads". Examples include SUGLETS® (by Colorcon) and comparable sugar spheres made by Dhanraj Sugars PVT. Ltd. and Emilio Castelli S.a.s. (see www.kingsresearch.com / blog / enhancing- drug-delivery-with-sugar-spheres). Optionally, a protective and / or delayed-release outer polymer layer may be added.

[0039] The composition is preferably contained within a pharmaceutical grade capsule. If necessary, the capsule may be an enteric coated capsule. Such capsules are well-known in the field. An example of suitable capsules are DRcaps® capsules (Lonza). DRcaps® capsules are composed of hypromellose as a structural polymer and may contain additives, opacifiers and / or colorants as appropriate. Each ingredients in DRcaps® capsules meets applicable pharmaceutical and / or food grade specifications. The table below provides a general composition overview of the Capsugel® DRcaps® capsule product.Capsugel® DRcaps® capsules compositionTypicalComponent Function Amount Regulatory ReferenceHypromellose Structure > 85% Pharmaceutical: Ph. Eur, USP-(HPMC) NF, JP, ChPFood: (EU) 231 / 2012 (E464),FCC, JSFA, JECFAGellan gum Acid resistance 5% Pharmaceutical: USP-NFFood: (EU) 231 / 2012 (E418), 21 CFR §172.665, FCC, JSFA, JECFA, GBColorants / opacifiers Color and / or < 7% Varies based on colorants(as applicable) opacity selected. Applicable food(product and / or pharmacopoeia identification) regulatory standards of the target markets and countries.Ink (as applicable) Imprinting < 150 Varies based on inks selected,(product pg / capsule Inks include food and / or identification) pharmaceutical grade constituents.Processing aids No functionality Amounts in Varies based on processing aids in empty hard accordance used and region produced, capsule. Used with and not Relevant pharmacopoeia to fulfill exceeding and / or food grade standards, manufacturing Good process. ManufacturingPractices to accomplish the intended purposePh. Eur: European Pharmacopoeia; USP-NF: United States Pharmacopeia - National Formulary; JP: Japanese Pharmacopoeia; ChP: Chinese Pharmacopoeia; EU: European Union; 21 CFR: United States Food and Drug Administration; FCC: Food Chemicals Codex; JSFA: Japanese Specifications for Food Additives; JECFA: Joint FAO / WHO Expert Committee on Food Additives; GB: Chinese Guobiao Standards

[0040] These compositions provide a suitable method of preventing or treating a Vibrio infection in a subject, in particular a Vibrio cholerae infection, by administering apharmaceutically effective amount of the composition according to any one of claims 1 to 12 to a subject in need thereof.

[0041] The following examples serve to illustrate certain aspects of the disclosure and should not be construed as limiting the claims. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.EXAMPLESComposition of Products

[0042] Spray Dried Powder

[0043] Fluidized Bed Coated BeadsSpray Drying

[0044] Formulations containing sugar and / or polymer and a buffered bacteriophage solution were pumped through a two-fluid nozzle and atomized into a drying chamber forming fine droplets. Upon atomization into the chamber, the fine droplets contact a heated co-current gas stream, evaporating solvent to yield solid particles. The solid particles were separated from the gas stream and collected.

[0045] Process parameters: Drying gas rate: 0.50 m3 / min; Liquid feed rate: 2.6 mL / min; Solids loading: 14 wt %; Atomization pressure: 1 bar; Spray dryer outlet temperature: 39 - 44 °C; Predicted relative humidity at outlet of spray dryer: 6%; Moisture content of spray dried phages (SDPs): = 1 - 6.5 wt %. Specifically, sugar and / or polymer excipients were added to buffered bacteriophage solutions to comprise ~ 90 wt % of total solids, with the remaining 10 wt % being buffer salts and bacteriophages. The device used was a custom- built open-loop unit with process air flow up to 45 kg / h, atomization pressures up to 6 barg, and inlet temperatures up to 100 deg C.Fluidized Bed Coating

[0046] Formulations containing sugar and / or polymer and a buffered bacteriophage solution were pumped through a nozzle and atomized into a fluid bed chamber. Upon atomization into the chamber, the droplets contact fluidized beads rising through a Wurster column, and then dry from the heated gas in the chamber as the beads fall back to the bed and repeat the cycle. The starting size of the bead core, the solids loading of the coating solution, and the coating time determine the thickness of the layers on the beads. Barrier layers containing polymer (HPMC-E3) with no phage were applied to protect the phage layer. An enteric polymer coating (Eudragit L30 D-55 or FL30 D-55) was used to provide protection of beads in environments below pH 5.5.

[0047] Process parameters: Airflow: 35-40 m3 / h; Atomization pressure: 1.5-1.7 bar;Solution flow rate: 0.5 - 1 mL / min; Inlet temperature: 55 - 65 °C; Core: Suglets, 710-850 pm. The device used was a M i n i-G latt / M id i-Glatt rated for batch sizes of 5 - 300 g, with process air flow range of 30 - 60 m3 / h, inlet temperatures up to 80 °C, and atomization pressures up to 6 barg.

[0048] The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.Stability Studies

[0049] Stability studies were conducted to assess formulations for protecting phages from heat and humidity. Several combinations of excipients with different ratios were tested for up to 1 years at three different temperatures and two humidity levels. Phages were mixed with each formulation, and the mixtures were spray dried. Sealed vials containing spray- dried powder were stored at 25 °C, 40 °C, 60 °C. Vials were opened at 25 °C with 60% relative humidity.

[0050] Figures 1 - 6 show results of stability testing. The formulations with sucrose or trehalose mixed with HPC at 50:50 ratio kept titer of all three phages from the initial titer at 25 °C for 1 year and at 40 °C for 6 months. These results indicated that those formulations protect phages at higher temperature (40 °C), which has not been previously reported. When tested the potency with animal model, these formulations kept well.In vivo studies

[0051] Phage ratios in composition: Each infant mouse was dosed with total 5 x 106PFU of ICP cocktails, which have ratio of ICP1, ICP2, and ICP3 as 1:1:1 or 10:1:1 or 1:10:1, before infection with 5 x 105CFU V. cholerae clinical strain. Twenty-four hours after infection, mice were killed and the V. cholerae surviving in the small intestine enumerated as CFU per smallintestine. Significance was calculated using the ANOVA test with the Tukey's post-hoc multiple comparisons test. Results are shown in FIG. 7.

[0052] Spray-dried PVC versus non-processed PVC: Each infant mouse was dosed with total 5 x 106PFU of ICP cocktails, which have ratio of ICP1, ICP2, and ICP3 as 1:1:1 before infection with 5 x 105CFU V. cholerae clinical strain. Spray-dried PVC was reconstituted with sterilized distilled water. Twenty-four hours after infection, mice were killed and the V. cholerae surviving in the small intestine enumerated as CFU per small intestine. Significance was calculated using the Kruskal-Wallis test with the Dunn's post-hoc multiple comparisons test. Results are shown in FIG. 8.

Claims

CLAIMSWhat is claimed is:

1. A composition, comprising: a. Vibrio bacteriophages ICP1, ICP2, and ICP3; and b. an excipient coating disposed on the bacteriophages, wherein the coating comprises hydroxypropyl P-cyclodextrin, HPC, HPMC, HPMC-AS, or PVP-VA.

2. The composition of claim 1, wherein each of the Vibrio bacteriophages represents 8% to 85% of the total amount of phage in the composition.

3. The composition of claim 2, wherein the Vibrio bacteriophages are present in substantially equal amounts.

4. The composition of any one of claims 1 to 3, wherein the excipient coating further comprises a sugar.

5. The composition of claim 4, wherein the sugar is trehalose, sucrose, or mannitol.

6. The composition of any one of claims 1 to 5, wherein the excipient coating further comprises an amino acid.

7. The composition of claim 6, wherein the amino acid is leucine.

8. The composition of any one of claims 1 to 7, wherein the excipient coating comprises hydroxypropyl P-cyclodextrin or HPC.

9. The composition of any one of claims 1 to 8, wherein the excipient coating comprises hydroxypropyl -cyclodextrin and trehalose.

10. The composition of any one of claims 1 to 9, wherein the sugar is present in a ratio of from 0:100 to 90:10 sugar: hydroxypropyl 0-cyclodextrin, HPC, HPMC, HPMC-AS, or PVP-VA.

11. The composition of claim 10, wherein the sugar is present in a ratio of from 45:55 to 55:45 sugar: hydroxypropyl P-cyclodextrin, HPC, HPMC, HPMC-AS, or PVP-VA.

12. The composition of any one of claims 1 to 11, further comprising an enteric coating material disposed on the excipient coating.

13. The composition of any one of claims 1 to 12, wherein the composition comprises solid particles made by spray-drying an aqueous suspension of the composition of the Vibrio bacteriophages and the excipients.

14. The composition of any one of claims 1 to 12, wherein the composition is made by a fluidized bed coating process, said process comprising atomizing a solution of the Vibrio bacteriophages into a Wurster column, then introducing the excipients into the Wurster column and Wurster coating the bacteriophages with the excipient coating, and optionally adding a protective and / or delayed-release outer polymer layer.

15. The composition of any one of claims 1 to 14, contained within a pharmaceutical grade capsule, optionally an enteric coated capsule.

16. A composition according to any one of claims 1 to 15 for use in preventing or treating a Vibrio infection by administering an effective amount of the composition to a subject in need thereof.

17. The composition of claim 16, wherein the Vibrio infection is a Vibrio cholerae infection.

Images