Patent Application
20230398178
This application incorporates by reference the Sequence Listing contained in the following eXtensible Markup Language (XML) file being submitted concurrently herewith:
Bacterial vaginosis is an imbalance in the vaginal microbiota that affects up to a third of people with vaginas throughout the world (
The current standard treatment for BV is oral or intravaginally applied antibiotics, which effectively clear out the potentially pathogenic anerobic bacteria characteristic of CSTIII and CSTIV communities; unfortunately, these broad-spectrum antibiotic treatments also target beneficial commensal Lactobacilli.5 As many as 30% of patients who undergo standard antibiotic treatments have a recurrence of BV,26 and the diminishment of beneficial Lactobacilli leaves patients vulnerable to secondary infections like vulvovaginal candidiasis. Novel methods of treatment, which involve introducing new strains of probiotic Lactobacilli have been proposed.27 A small five-person pilot study of vaginal microbiota transplants found that treating BV patients with antibiotics and then inoculating them with strains from healthy donors was able to lead to lasting conversion to CSTI communities in four of five patients.28 While these treatments showed great promise, they were not successful for all patients in the cohort, indicating that further interventions may be needed.
This Summary introduces a selection of concepts in simplified form that are described further below in the Detailed Description. This Summary neither identifies key or essential features, nor limits the scope, of the claimed subject matter.
Described herein is a method of treating or preventing a vaginal infection of Gardnerella vaginalis in a subject. The method can include administering to the subject an effective amount of a composition comprising one or more of galactose, D-fucose, or mucin 5B. In some instances, the composition includes galactose. In some instances, the composition includes D-fucose. In some instances, the composition includes mucin 5B. In any of the foregoing methods, the galactose, N-acetyl D-galactosamine, N-acetyl D-glucosamine, D-fucose, or mucin 5B can administered in an amount from 0.1 wt % to 50 wt %.
Described herein is a method of treating or preventing a vaginal infection of Gardnerella vaginalis in a subject. The method can include administering to the subject an effective amount of a composition comprising one or more of galactose, N-acetyl D-galactosamine, N-acetyl D-glucosamine, D-fucose, or mucin 5B bonded to a surface. In some instances, galactose bonded to the surface. In some instances, N-acetyl D-galactosamine bonded to the surface. In some instances, N-acetyl D-glucosamine bonded to the surface. In some instances, D-fucose bonded to the surface. In some instances, mucin 5B bonded to the surface. In any of the foregoing methods, the surface can be a polymer, a silk fibroin (SF), a norbornene polymer, a star polymer, or a dendrimer. In any of the foregoing methods, the galactose, D-fucose, or mucin 5B can be administered in an amount from 0.1 wt % to 50 wt %.
In any of the foregoing methods, the method can reduce the concentration of Gardnerella vaginalis in the vagina of the subject. The method can reduce expression of vaginolysin by Gardnerella vaginalis in the vagina of the subject. The method can reduce concentration of vaginolysin in the vagina of the subject. The method can reduce mortality rates for endocervical cells of the subject. The method can reduce biofilm formation caused by the vaginal infection of Gardnerella vaginalis. The method can treat the vaginal infection of Gardnerella vaginalis. The method can prevent the vaginal infection of Gardnerella vaginalis. The subject can have bacterial vaginosis. In some instances, the subject has previously had at least three bacterial vaginosis infections.
Any of the foregoing methods can include diagnosing the subject with the vaginal infection of Gardnerella vaginalis.
In any of the foregoing methods, the composition can be formulated for topical or vaginal administration.
Any of the foregoing methods can include administering a Lactobacillus probiotic to the subject.
Any of the foregoing method can include administering an antibiotic, such as metronidazole, to the subject.
The following Detailed Description references the accompanying drawings that form a part this application, and which show, by way of illustration, specific example implementations. Other implementations may be made without departing from the scope of the disclosure.
The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.
A description of example embodiments follows.
Reference numbers in superscripts herein refer to the corresponding literature listed in the attached Bibliography which forms a part of this Specification, and the literature is incorporated by reference herein.
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly-used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and/or as otherwise defined herein.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
When introducing elements disclosed herein, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. Further, the one or more elements may be the same or different.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of, e.g., a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein, the term “comprising” can be substituted with the term “containing” or “including.”
As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the terms “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the disclosure, can in some embodiments, be replaced with the term “consisting of,” or “consisting essentially of” to vary scopes of the disclosure.
As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and/or.”
It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.
Compounds described herein include those described generally, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the relevant contents of which are incorporated herein by reference.
Unless specified otherwise within this specification, the nomenclature used in this specification generally follows the examples and rules stated in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979, which is incorporated by reference herein for its chemical structure names and rules on naming chemical structures. Optionally, a name of a compound may be generated using a chemical naming program (e.g., CHEMDRAW®, version 17.0.0.206, PerkinElmer Informatics, Inc.).
The phrase “pharmaceutically acceptable” means that the substance or composition the phrase modifies is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids, and suitable inorganic and organic bases.
Examples of pharmaceutically acceptable acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable acid addition salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxy-ethanesulfonate, hydroxymaleate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 2-phenoxybenzoate, phenylacetate, 3-phenylpropionate, phosphate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.
Either the mono-, di- or tri-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form.
Salts derived from appropriate bases include salts derived from inorganic bases, such as alkali metal, alkaline earth metal, and ammonium bases, and salts derived from aliphatic, alicyclic or aromatic organic amines, such as methylamine, trimethylamine and picoline, or N+((C1-C4)alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
Compounds described herein can also exist as “solvates” or “hydrates.” A “hydrate” is a compound that exists in a composition with one or more water molecules. A hydrate can include water in stoichiometric quantities, such as a monohydrate or a dihydrate, or can include water in random amounts. A “solvate” is similar to a hydrate, except that a solvent other than water, such as methanol, ethanol, dimethylformamide, diethyl ether, or the like replaces water. Mixtures of such solvates or hydrates can also be prepared. The source of such solvate or hydrate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
Compounds disclosed herein may exist as stereoisomers. For example, compounds disclosed herein may have asymmetric centers, chiral axes, and chiral planes (e.g., as described in: E. L. Eliel and S. H. Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, or as individual diastereomers or enantiomers.
“Pharmaceutically acceptable carrier” refers to a non-toxic carrier or excipient that does not destroy the pharmacological activity of the agent with which it is formulated and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent. Pharmaceutically acceptable carriers that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
“Treating” or “treatment,” as used herein, refers to taking steps to deliver a therapy to a subject, such as a mammal, in need thereof (e.g., as by administering to a mammal one or more therapeutic agents). “Treating” or “treatment” includes inhibiting the disease or condition (e.g., as by slowing or stopping its progression or causing regression of the disease or condition), and relieving the symptoms resulting from the disease or condition. The term “treating” or “treatment” refers to the medical management of a subject with the intent to improve, ameliorate, stabilize (i.e., not worsen), prevent or cure a disease, pathological condition, or disorder—such as the particular indications exemplified herein. This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive treatment (treatment employed to supplement another therapy). Treatment also includes diminishment of the extent of the disease or condition; preventing spread of the disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
“Administering” or “administration,” as used herein, refers to providing a compound, composition, or pharmaceutically acceptable salt thereof described herein to a subject in need of treatment or prevention.
“A therapeutically effective amount” or “an effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or biological result (e.g., treatment, healing, inhibition or amelioration of physiological response or condition, etc.). Non-limiting examples of desired therapeutic or biological results include disruption of biofilm formation, growth, and/or maintenance, for example, at or proximate to the surface of an implanted medical device. Effective reductions of signs and/or symptoms associated with infection can be determined by one or more suitable means in the art.
The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. A therapeutically effective amount may vary according to factors such as disease state, age, sex, and weight of an individual, e.g., a mammal, mode of administration and the ability of a therapeutic, or combination of therapeutics, to elicit a desired response in an individual.
An effective amount of an agent to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art. For example, suitable dosages can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, or from about 0.01 mg/kg to about 1 mg/kg body weight per treatment. Determining the dosage for a particular agent, subject and disease is well within the abilities of one of skill in the art. Preferably, the dosage does not cause or produces minimal adverse side effects.
In one aspect, the disclosure provides methods of treating or preventing bacterial vaginosis in a subject in need thereof. The method can include administering to the subject an effective amount of galactose (Gal), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), or D-fucose (D-Fuc), or a pharmaceutically acceptable salt of any thereof.
In some embodiments, the subject has a vaginal infection of Gardnerella vaginalis.
“Virulence,” as used herein, refers to a phenotypic state of Gardnerella vaginalis associated with infection that may harm its host, for example, the host's endocervical cells. In some embodiments, the method reduces the quantity of a population of Gardnerella vaginalis in the vagina, reduces mortality rates for endocervical cells, reduces expression of vaginolysin in the vagina, or a combination thereof.
In some embodiments, a method disclosed herein reduces expression of vaginolysin in the vagina by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, a method reduces expression of vaginolysin in the vagina by at least about 30%.
Expression of vaginolysin in the vagina can be determined by a person of ordinary skill using methods known in the art, for example, by quantitative reverse transcription polymerase chain reaction (RT-qPCR), or, alternatively, at the RNA level using RNA sequencing (RNA-Seq) or a microarray.
In some embodiments, attenuating virulence of Gardnerella vaginalis includes inhibiting or reducing biofilm formation. “Biofilm,” as used herein, refers to a structured community of microbial cells that is adherent to a surface. In some embodiments, a method reduces biofilm formation by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, a method reduces biofilm formation by at least about 30%.
In another aspect, described herein are methods of inhibiting formation of a biofilm on a surface. The methods include contacting the surface with a monosaccharide (e.g., a therapeutically effective amount of a monosaccharide). The methods can also include contacting the surface with a composition that includes a glycopolymer of a monosaccharide (e.g., a therapeutically effective amount of a composition that includes a glycopolymer of a monosaccharide). The monosaccharide can be galactose (Gal), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), or D-fucose (D-Fuc). In some instances, the method can include contact the surface with mucin 5B (MUC5B).
As used herein, “subject” includes humans, domestic animals, such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.) and livestock (e.g., pigs, cattle, sheep, goats, horses, etc.), and non-domestic animals. In some embodiments, a subject is a human.
“Subject in need thereof,” as used herein, refers to a subject (e.g., a mammalian subject such as a human) diagnosed with or suspected of having a vaginal infection of Gardnerella vaginalis. “Subject in need thereof” includes those subjects who already have the undesired physiological change or disease as well as those subjects prone to have the physiological change or disease. “Subject in need thereof” can also refer to a subject diagnosed with or suspected of having bacterial vaginosis.
The administration of the compounds (agents, salts, etc.) and compositions typically occurs by topical or vaginal administration at or near the site of infection.
In some embodiments, a composition is provided in a liquid form. In some embodiments, a composition comprises a dose of from about 0.1 g/liter to about 50 g/liter, for example, about: 0.1 g/liter, 0.2 g/liter, 0.3 g/liter, 0.4 g/liter, 0.5 g/liter, 0.6 g/liter, 0.7 g/liter, 0.8 g/liter, 0.9 g/liter, 1 g/liter, 2 g/liter, 3 g/liter, 4 g/liter, 5 g/liter, 6 g/liter, 7 g/liter, 8 g/liter, 9 g/liter, 10 g/liter, 11 g/liter, 12 g/liter, 13 g/liter, 14 g/liter, 15 g/liter, 16 g/liter, 17 g/liter, 18 g/liter, 19 g/liter, 20 g/liter, 21 g/liter, 22 g/liter, 23 g/liter, 24 g/liter, 25 g/liter, 26 g/liter, 27 g/liter, 28 g/liter, 29 g/liter, 30 g/liter, 31 g/liter, 32 g/liter, 33 g/liter, 34 g/liter, 35 g/liter, 36 g/liter, 37 g/liter, 38 g/liter, 39 g/liter, 40 g/liter, 41 g/liter, 42 g/liter, 43 g/liter, 44 g/liter, 45 g/liter, 46 g/liter, 47 g/liter, 48 g/liter, 49 g/liter, 50 g/liter, 60 g/liter, 70 g/liter, 80 g/liter, 90 g/liter, 100 g/liter, 150 g/liter, 200 g/liter, 250 g/liter, 300 g/liter, 350 g/liter, 400 g/liter, 450 g/liter, or 500 g/liter.
In some embodiments, a composition comprises a dose of from about 0.1 wt % to 50 wt %, for example about 0.1 wt %, 0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 0.6 wt %, 0.7 wt %, 0.8 wt %, 0.9 wt %, 1 wt %, 2 wt %, 3 wt %, 4 wt %, 5 wt %, 6 wt %, 7 wt %, 8 wt %, 9 wt %, 10 wt %, 11 wt %, 12 wt %, 13 wt %, 14 wt %, 15 wt %, 16 wt %, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt %, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt %, 33 wt %, 34 wt %, 35 wt %, 36 wt %, 37 wt %, 38 wt %, 39 wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %, 45 wt %, 46 wt %, 47 wt %, 48 wt %, 49 wt %, or 50 wt %. In some embodiments, the composition comprises a dose of from about 0.1 wt % to 10 wt %. In some embodiments, the composition comprises a dose of from about 0.1 wt % to 5 wt %. In some embodiments, the composition comprises a dose of from about 0.1 wt % to 3 wt %. In some embodiments, the composition comprises a dose of from about 0.1 wt % to 2 wt %. In some embodiments, the composition comprises a dose of from about 0.1 wt % to 1 wt %. In some embodiments, the composition comprises a dose of from about 0.1 wt % to 0.5 wt %. In some embodiments, the composition comprises a dose of from about 0.5 wt % to 10 wt %. In some embodiments, the composition comprises a dose of from about 0.5 wt % to 5 wt %. In some embodiments, the composition comprises a dose of from about 0.5 wt % to 2 wt %. In some embodiments, the composition comprises a dose of from about 0.5 wt % to 1 wt %.
In some embodiments, a composition is provided in a dried form.
In some embodiments, a composition is provided in a gel form.
Administration of a compound, composition, or pharmaceutically acceptable salt described herein may be in conjunction with another active ingredient (e.g., an antibiotic, such as metronidazole), for example, simultaneously in the same composition, simultaneously in different dosage forms, or sequentially. A compound, composition, or pharmaceutically acceptable salt described herein and another active ingredient may be formulated in a single combination, multiple combinations, or separate compositions.
The other active ingredient (e.g., an antibiotic, such as metronidazole) can be administered in any suitable manner, e.g., by parenteral or nonparenteral administration, including by aerosol inhalation, injection, infusions, ingestion, transfusion, implantation or transplantation. For example, the other active ingredient described herein may be administered to a subject trans-arterially, intradermally, subcutaneously, intratumorally, by intramedullar administration, intranodally, intramuscularly, intravenously (e.g., through an IV drip or by intravenous (i.v.) injection), intranasally, intrathecally or intraperitoneally. In some embodiments, the administration is intravenous. In some embodiments, the administration is topical. In some embodiments, the administration is oral. In some embodiments, the administration is by injection, for instance, directly into a tissue, organ, or site of infection. In some embodiments, the administration is ex vivo. In some embodiments, other active ingredient is administered by routes such as oral, endobronchial, intrathecal, intracisternal, intra-articular, intraperitoneal, ophthalmic (e.g., in an ophthalmic preparation such as eye drops, intraocular injections, ointments), aerosol, irrigant, peritoneal lavage, endobronchial and intrathecal administration.
In some embodiments, the other active ingredient is administered topically, orally, intravenously, nasally, ocularly, or transdermally. In some embodiments, the other active ingredient is administered topically. In some embodiments, the other active ingredient is administered orally. In some embodiments, the other active ingredient is administered intravenously. In some embodiments, the other active ingredient is administered nasally. In some embodiments, the other active ingredient is administered ocularly. In some embodiments, the other active ingredient is administered transdermally.
In some embodiments, an amount (e.g., a therapeutically effective amount) of a compound or pharmaceutically acceptable salt thereof is sufficient to increase a rate of clearance of a biofilm in a subject, for example, compared to the same subject were it left untreated. In some embodiments, an amount (e.g., a therapeutically effective amount) of a compound or pharmaceutically acceptable salt thereof is sufficient to increase the rate of clearance of a biofilm by at least about 20%, for example, by at least about: 50%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold.
In some embodiments, an amount (e.g., a therapeutically effective amount) of a compound or pharmaceutically acceptable salt thereof is sufficient to reduce the quantity of a population of Gardnerella vaginalis in the vagina by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, an amount (e.g., a therapeutically effective amount) of a compound or pharmaceutically acceptable salt thereof is sufficient to reduce the quantity of a population of Gardnerella vaginalis in the vagina by at least about 30%.
In some embodiments, a monosaccharide is incorporated into a formulation for therapeutic administration (e.g., a pharmaceutical composition). In some embodiments, a composition comprising a glycopolymer of a monosaccharide is incorporated into a formulation for therapeutic administration (e.g., a pharmaceutical composition).
In some embodiments, a pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or diluents. In some embodiments, a pharmaceutical composition further comprises one or more additional therapeutics, i.e., therapeutic agents (e.g., an antibiotic). Pharmaceutical compositions may be formulated into preparations in, for example, solid, semi-solid, liquid or gaseous forms, such as capsules, gels, granules, microspheres, ointments, powders, solutions, drops, and tablets. Defined, or semi-defined mucin glycan compositions may be formulated for various routes of administration, for example, oral formulations, intravenous formulations, or in the form of a douche. In some embodiments, a defined, or semi-defined mucin glycan composition is formulated into an ointment.
Without wishing to be bound by theory, soluble glycans may be metabolized, but those bonded to a polymer backbone are less likely to be metabolized. Binding the glycans to a surface concentrates the glycan and provides a medium for delivery the glycan to a particular location, namely, at or near the site of infection. Many different surfaces are suitable, including polymers (including bottlebrush polymers and star polymers) and dendrimers. The Exemplification describes experiments with two different types of polymers, a silk fibroin and a norbornene polymer, but a variety of polymers are suitable so long as they are non-toxic when administered as described herein.
Mucins are heavily O-glycosylated glycoproteins that are found in mucous secretions (secreted mucins) and on the cell surface (membrane-bound (transmembrane) mucins). Secreted mucins include gel-forming mucins and non-gel-forming (soluble) mucins.
Mucin genes are expressed in a tissue- and/or region-specific fashion, for example, in the airway, digestive system, reproductive system, and different regions of the gastrointestinal tract. About 20 different mucin genes have been cloned, including gel-forming mucin genes such as MUC2, MUC5AC, MUC5B, MUC6 and MUC19; soluble mucin genes such as MUC7, MUC8, MUC9 and MUC20; and transmembrane mucin genes such as MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15 and MUC21.
Non-limiting examples of mucin genes include human MUC1 (e.g., GenBank: AAA60019.1, UniProtKB/Swiss-Prot: P15941.3, Gene ID 4582), porcine MUC1 (e.g., NCBI: XP_020945387.1), human MUC2 (e.g., GenBank: AAB95295.1, Gene ID 4583), porcine MUC2 (e.g., NCBI: XP_020938243.1), human MUC5AC (e.g., GenBank: ABV02582.1, UniProtKB/Swiss-Prot: P98088.4, Gene ID 4586), porcine MUC5AC (e.g., NCBI: XP_020938242.1), human MUC5B (e.g., UniProtKB/Swiss-Prot: Q9HC84.3, Gene ID 727897), porcine MUC5B (e.g., NCBI: XP_020938146.1), human MUC6 (e.g., GenBank: AZL49144.1) and porcine MUC6 (e.g., NCBI: XP_020938133.1).
Additional non-limiting examples of mucin genes include MUC2 (e.g., HomoloGene 130504, 131905, 132025, or 133451), MUC5AC (e.g., UniGene IDs 3881294, 1370646, 1774723, 1133368, 441382, and 5878683, HomoloGene 130646; Gene ID 100170143; and reference sequences AAC48526, AAD19833, and AAD19832), MUC5B (e.g., HomoloGene 124413), MUC6 (e.g., HomoloGene 18768), MUC19 (bovine submaxillary mucin (BSM), e.g., Gene ID 100140959; HomoloGene 130967; and reference protein sequence XP_0035861 12.1).
A mucin protein comprises an amino region and/or a carboxy region that are cysteine-rich and a central region enriched in serine and/or threonine residues. Native mucin glycans are typically built upon an N-acetylgalactosamine that is O-linked via its C-1 hydroxyl to serine or threonine residues of a mucin protein. The monosaccharide unit or series of monosaccharide units that, in a native mucin glycan, would be O-linked via the C-1 hydroxyl of the monosaccharide unit or first monosaccharide unit in the series of monosaccharide units, respectively, to a serine or threonine residue of the mucin protein is also referred to herein as the “glycan core” or “mucin glycan core.”
G. vaginalis is one bacterial species that has been heavily implicated as an initiator of severe bacterial vaginosis. This microbe has two main phenotypic behaviors that contribute to its virulence: its ability to form biofilms that allow it to persistently colonize the vaginal epithelium,29 and the secretion of the cholesterol-dependent cytolysin vaginolysin, which lyses host epithelia, leading to inflammation.30,31 At the same time, this organism is found in the vaginas of symptomless patients,24,32 suggesting that it is capable of being domesticated in the right conditions. This led us to hypothesize that factors in host mucus may cause G. vaginalis to suppress its virulent behaviors.
Cervical mucus is formed of the mucin MUC5B, a long protein polymer. MUC5B is a glycoprotein, meaning that it is post translationally modified by the addition of sugars, which contribute up to 80% of its molecular weight. Individual mucin samples can contain dozens of glycan structures, built from the same 5 monosaccharide sugars: D-galactose (Gal), N-acetyl D-galactosamine (GalNAc), N-acetyl D-glucosamine (GlcNAc), L-fucose (L-Fuc), and N-acetyl D-neuraminic acid (NeuNAc, or sialic acid). Since it is difficult to get large quantities of cervical mucus for our studies, we instead isolated MUC5B from fresh saliva, which is highly similar to MUC5B expressed in the vaginal tract.33 We then reconstituted the mucin into gels at higher concentrations, creating a model to study bacterial behavior.
These mucin proteins have many functions in modulating the microbial behavior.34 Canonically, they are a physical barrier, which helps them prevent bacteria from reaching the epithelial surface, and can spatially distribute microbes, creating different niches.35 Secondly, mucin glycans can be a source of diverse and complex nutrients, which can metabolically shape the microbiota.36 Glycans may select for beneficial microbes which produce specific glycosidases, as well as facilitate cooperation across species which produce complementary degradative enzymes. Recently, a third function of mucins has been discovered, which is as an active regulator of microbial virulence. In this model, microbes are able to sense and respond to the glycan structures on healthy mucus.37 Mucin and mucin glycans have non-nutritive effects on several opportunistic pathogens across kingdoms, where the transcriptomes of these microbes change in response to healthy mucin, resulting in decreases of biofilm formation, toxin production, quorum sensing, hyphae formation, and other virulence factors.38-40 We therefore hypothesize that healthy mucin environments could play a role in preventing pathogen virulence and outgrowth in the vagina.
Here, we studied the influence of healthy vaginal mucin on Gardnerella vaginalis, in order to understand more about how natural host defense mechanisms alter the behavior of opportunistic microbial pathogens, and to leverage this understanding to create interventions that could potentially treat BV without harming commensal Lactobacilli.
To begin, we cultured G. vaginalis 14018 biofilms in NYCIII media anerobic conditions in the presence or absence of a 0.2 wt % MUC5B mucin gel (
This opens the possibility that mucin's glycans may be responsible for its influence on bacterial behavior. To probe this, we tested whether the monosaccharides that compose MUC5B's complex glycans influenced G. vaginalis biofilm formation (
Next, we sought to determine whether these effects were unique to the 14018 strain of G. vaginalis. Thus, we compared against another strain, Gardnerella vaginalis 49145, which was isolated from a patient with bacterial vaginosis. We observed that 49145 showed a similar pattern to 14018, with a reduction of biofilm formation in the presence of MUC5B, galactose, and D-fucose (
Given that mucin and D-fucose are able to suppress biofilm formation by G. vaginalis 49145 and 14018, we next wondered whether this effect translated into reduced virulence against epithelial cells. To test this, we used an endocervical cell line model (End1), introduced these strains as a pathogen challenge, and then measured the viability of the endocervical cells after 24 hours (
Since the barrier function of mucin does not seem sufficient for its effects, we expect that mucin may be acting as a biochemical signal to reduce virulence. We used RT-qPCR to monitor the expression of vaginolysin (vly), a cholesterol-dependent cytolysin that kills epithelial cells and is a major contributor to Gardnerella virulence (
We next sought to determine if grafting galactose onto a polymer backbone could enhance its effects. While 0.2 wt % of soluble galactose inhibited growth in glucose rich media by 90%, when galactose was tethered onto silk fibroin, treatment with 2.5 wt % SF(S)-Gal reduced growth by 99.7%, an improvement of 2-3 orders of magnitude (
Finally, we tested whether the silk glycopolymers impacted the growth of a commensal bacterium Lactobacillus crispatus, which is a lead candidate for most vaginal probiotics. Here, we observed that none of the silk glycopolymers had a detrimental effect on the growth of strain JV001 (
We discovered that MUC5B mucin, its associated monosaccharide galactose (=D-Gal) and an unnatural analogue D-fucose (=D-Fuc) are able to robustly dissolve pre-established Gardnerella vaginalis biofilms. The observed reduction constitutes a 10-15-fold decrease in the number of colony-forming units in the biofilm fraction (
We hypothesized that the biofilm disruption activity of mucin glycan Gal and its analogue D-Fuc has the potential to increase the susceptibility of Gardnerella vaginalis to antibiotic metronidazole. For this we performed a minimum biofilm eradication assay wherein established biofilms were exposed to metronidazole alone, glycans alone or the combination thereof. Across several metronidazole concentrations we observe a synergistic effect between the antibiotic and monosaccharides, leading to a significant increase in the magnitude of the biofilm disruption achieved in the combined conditions.
The vaginal canal is coated with the mucin MUC5B and is colonized by a variety of commensal Lactobacilli. Here, we targeted the Gardnerella vaginalis, an opportunistic pathogen that can contribute to symptomatic bacterial vaginosis, but is carried asymptomatically in some patients. We hypothesized that mucin may alter pathogenic behaviors of G. vaginalis, and indeed, exposure to MUC5B limited its biofilm formation, toxin production, and killing of endocervical cells. Next, we identified that galactose and a structurally similar monosaccharide, D-fucose, were able to reproduce mucin's virulence suppression. Additionally, these molecules limited G. vaginalis growth in glucose-rich media, suggesting that they may function by rerouting microbial metabolism. Finally, we saw that when galactose was grafted onto silk fibroin polymers (SF-Gal), it was extremely toxic to G. vaginalis, but not L. crispatus. Additionally the Galactose tethered to a norbornene backbone had a similar effect, as did SF-GalNAc and SF-GlcNAc. Together, these results suggest that mucin and galactose play an important role in maintaining health in the vaginal microbiome.
Here, we showed that mucin, galactose, and the galactose-similar molecule D-fucose are able to inhibit biofilm formation by the vaginal pathogen G. vaginalis. Further, galactose suppresses bacteria growth by one log in glucose-rich media, an effect that is amplified by several orders of magnitude when galactose is grafted onto a polymer backbone. The mechanism by which these antimicrobial polymers function is still unclear, but we hypothesize that they interfere with the efficient transport or utilization of nutrients, or they disrupt membrane integrity.
This study demonstrated that both natural mucin and synthetic glycopolymers have a profound effect on bacterial virulence. This illustrates the importance of mucin as a natural protective mechanism the host uses to disrupt bacterial virulence. On the other hand, the galactose-based synthetic polymers appear as a novel method for inhibiting bacterial growth. Most antimicrobial polymers are highly positively charged, or zwitterionic, and act on broad categories of bacteria by disrupting their naturally negatively charged membranes.51 These polymers appear to act by an entirely orthogonal mechanism that enables them to be selective, as demonstrated by their impotence against Lactobacillus crispatus. These materials provide a case study for the development of mucin-inspired antimicrobial agents that can selectively target pathogenic organisms while leaving beneficial commensals intact. Such therapies could provide a new paradigm of treating infections, complementing vaginal microbiome transplants by aiding in the reduction of pathogenic bacteria when probiotic strains are introduced.
Here, we explored one method of creating mucin-inspired glycan-bearing materials: a bottlebrush structure on a polymer backbone. However, we expect that there is significant space for exploring alternative architectures, such as surfaces or thicker gels. We observed that grafting the same sugar onto either silk fibroin or norbornene resulted in a bioactive polymer, indicating that the specific backbone identity is not necessarily essential for function; rather, it seems that the act of being grafted increases sugar activity. Additionally, materials which blend different bioactive sugar monomers could be created, enabling the simultaneous targeting of multiple microbial phenotypes.
With the rise of clinical antimicrobial resistance, these materials, which treat infections by hijacking nutrient sensing pathways, rather than directly killing bacteria, present a novel strategy for treating infections that can target pathogens while leaving the commensal microbiome intact.
The bacterial strains Gardnerella vaginalis ATCC 14018 and ATCC 49145 were obtained from ATCC and were originally isolated from people with active bacterial vaginosis. JCP7275 was obtained from BEI Resources. Bacteria were cultivated in modified ATCC NYCIII media or brain-heart infusion (BHI; BD 237500) 1.5% agar plates at 37° C. in airtight containers with Mitsubishi AnaeroPack-Anaero Anaerobic Gas Generators (Thermo Scientific; R681001), which consume oxygen and produce high concentrations of carbon dioxide. NYCIII media was prepared in 2× concentrations, flash cooled, and thawed in an anaerobic chamber the day before use. Briefly, modified NYCIII media was prepared in 250 ml batches in two steps. First, 2 g HEPES, 7.5 g Proteose Peptone No. 3 (BD 211693), 2.5 g NaCl, and 177.5 ml miliQ-distilled water were mixed, adjusted to pH 7.3, and autoclaved for 15 minutes. When the solution had cooled to room temperature, we added 12.5 ml of yeast extract solution (prepared by dissolving wt % Bacto Yeast Extract in miliQ-distilled water and sterilizing with a 0.2 μm syringe filter), ml of heat-inactivated horse serum (Millipore Sigma; H1138), 10 ml of 50 wt % 0.2 μm sterile filtered glucose solution. Media was then aliquoted into 10 ml tubes, flash cooled in liquid nitrogen, and stored at −80° C.
Mucin Purification from Pooled Saliva
Submandibular saliva was collected from informed consenting volunteers as described previously.53 Saliva donations from five donors were pooled before MUC5B was purified using liquid chromatography.40 Purified MUC5B was flash cooled, lyophilized, and stored at −80° C. Before use, MUC5B was rehydrated in Milli-Q-purified water and agitated at 4° C. overnight. Protocols involving samples from human participants were approved by the Massachusetts Institute of Technology's Committee on the Use of Humans as Experimental Subjects.
Overnight cultures of G. vaginalis were inoculated from glycerol stocks and grown for 12-24 hours in NYCIII media at 37° C. in an anaerobic chamber. Biofilms were prepared by diluting overnight cultures 1:20 into 50-100 μl of NYCIII media and any additives in a 96-well polystyrene plate for a starting inoculum of OD600˜0.1 or 105-106 CFU per ml. The cultures were then incubated statically for 24 hours at 37° C. in an anaerobic chamber.
To measure total growth and relative biofilm formation, the number of cells in the biofilm and in suspension were counted using a colony forming unit (CFU) assay as previously described. 7 Briefly, the supernatant was removed and transferred to a new 96-well plate, the biofilm was washed twice with 100 μl of phosphate-buffered saline (PBS), and the washes were added to the supernatant. The biofilm was then detached and resuspended in 100 μl of PBS by vigorously scraping with a pipette tip for 30 s. Each culture fraction was then serially diluted and plated on BHI agar. Colonies were counted after 48 hours of growth at 37° C. in an anaerobic chamber. The fraction biofilm was calculated as (biofilm CFU per ml)/(biofilm CFU per ml+supernatant CFU per ml). The total growth was calculated as biofilm CFU per ml+supernatant CFU per ml. All of the conditions had at least three biological replicates (individual data points shown on graphs).
A table of the primers used in this study is provided in Table 1 (SEQ ID NOS: 1-4). Gene expression analysis was performed using quantitative PCR with reverse transcription (RT-qPCR) as previously described. 40 Briefly, subcultures were grown as described above, but in PCR tubes. After 3 hours of growth, cells were centrifuged, the supernatant was removed, and the pellet was flash cooled in liquid nitrogen. Total nucleic acids were then extracted using the MasterPure Complete RNA Purification kit (Lucigen). Genomic DNA was removed using the Turbo DNA-free kit (Ambion). Total RNA was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies) and stored at −80° C. cDNA was synthesized with the Protoscript II First-Strand cDNA Synthesis kit (New England Biolabs). About 2 ng of cDNA was used as a template for qPCR using SYBR Green Master Mix (Thermo Fisher Scientific) performed using the Cycler 480 II Real-time PCR Machine (Roche). Gene expression changes were calculated as the mean change in qPCR cycle threshold compared to 16S rRNA (ΔCt) and are reported as log2[fold change]=ΔCt_media−ΔCt_sample. Each sample was analyzed with at least three technical replicates.
To identify Gardnerella strains lacking vaginolysin (vly), the vly sequence was blasted to the vaginal genomes of Gardnerella strains available through BEI. Strains that did not yield a blast result were grown for microbial gDNA extraction (Qiagen) and tested against the G. vaginalis ATCC49145 strain encoding vly. The presence of vly was determined by PCR amplification of the flanking regions using primers obtained from Cerca 2015. The PCR products were analyzed by gel electrophoresis and sequences by Genewiz to confirm the sequence identity.
The norbornene polymers were synthesized and characterized as described in Kruger et. al. 2021.42 The experiments described utilized cis polymers of 200 units in length and 20% galactose grafting density. Experiments used polymers at 2 wt %.
The overall synthesis of the galactose norbornene polymers was completed as illustrated in Scheme 1. The synthesis of the requisite reactants was completed as follows.
Known compound 4 was synthesized according to a modified procedure for compound 37 in Percec et. al. J. Am. Chem. Soc. 2013. To a 500 mL round bottom flask equipped with a stir bar 5.06 g of protected galactoside S3 (7.21 mmol, 1 equiv.) was added. In a 24 mL vial a solution of 1 M NaOMe in MeOH was freshly prepared by addition of 230 mg sodium metal into 10 mL MeOH. This solution was used to basify at least 140 mL MeOH to pH 9-10 by pH paper. The protected galactoside S3 was dissolved in 130 mL of the pH 9-10 NaOMe/MeOH solution and 1.42 mL piperidine (14.4 mmol, 2 equiv.) was added. The solution was heated to 40° C. and stirred for 48 hours after which the reactant solution was a light champagne color. The reactant solution was concentrated and partitioned between 40 mL each H2O and EtOAc, and washed with a further 2×40 mL portions EtOAc. The aqueous phase was basified to pH 11 by pH paper with 1-3 g Amberlyst A26 hydroxide form resin and washed with 3×40 mL portions methylene chloride (note that the Amberlyst resin needed to be physically present within the separatory funnel to aid in piperidine removal). The aqueous phase was transferred to a 500 mL round bottom flask and the residual methylene chloride was removed under reduced pressure. The aqueous phase was passed over a 0.22 μm syringe filter, neutralized by pH paper with 1-10 mL 1 M HCl, and lyophilized. The product was obtained as a light yellow resinous crystalline solid. The product was generally obtained with residual piperidine contaminant and was extremely hygroscopic. Consequently, the mass of product generally exceeded that of a theoretical 100% yield, however, the material was still readily grafted in the desired stoichiometry to polymers without accounting for these impurities. No Rf was obtained since the product would not migrate by normal phase TLC in any solvent system tested and streaked so heavily on reverse phase TLC as to be impractical. Average Yield: ≥99%. Minimum Yield: 97%. Maximum Yield: ≥99%. 1H NMR (600 MHz, deuterium oxide) δ 4.35 (d, J=7.9 Hz, 1H), 4.02 (dt, J=11.4, 4.0 Hz, 1H), 3.85 (d, J=3.5 Hz, 1H), 3.80-3.74 (m, 1H), 3.74-3.64 (m, 12H), 3.62 (dd, J=7.8, 4.3 Hz, 1H), 3.58 (dd, J=9.9, 3.4 Hz, 1H), 3.46 (abq, J=9.9, 7.9 Hz, 1H).
Compound 1 was synthesized according to a modified procedure for compound 15-NHS in Werther, et al. Chem.—A Eur. J. 2017. To a flame dried N2 flushed 100 mL round bottom flask 2.0 g exo-norbornene-5-carboxylic acid S5 (14.5 mmol, 1 equiv.), 2.33 g N-hydroxysuccinimide (20.2 mmol, 1.4 equiv.), 3.05 g 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl (15.9 mmol, 1.1 equiv.) were all added and dissolved in 24.1 mL dry methylene chloride. The reaction was allowed to proceed overnight before concentrating. The reactant solution remained clear and colorless throughout the reaction. The concentrate was partitioned between 90 mL each EtOAc and H2O, and the aqueous phase was extracted with a further 2×90 mL portions EtOAc. The organic phases were combined and washed with 2×45 mL portions saturated NH4Cl, 2×90 mL portions saturated NaHCO3, 1×90 mL portion brine, dried over MgSO4, and concentrated. Product was obtained as an off-white solid with Rf=0.48 on 30% EtOAc:Hexanes. Average Yield: 80%. Minimum Yield: 73%. Maximum Yield: 88%. 1H NMR (600 MHz, chloroform-d) δ 6.23 (dd, J=5.7, 3.0 Hz, 1H), 6.17 (dd, J=5.7, 3.1 Hz, 1H), 3.32-3.24 (m, 1H), 3.02 (m, 1H), 2.86 (d, J=6.6 Hz, 5H), 2.53 (ddd, J=9.0, 4.5, 1.4 Hz, 1H), 2.07 (dt, J=12.0, 3.8 Hz, 1H).
The norbornene-galactose polymer (“Cis 200mer”) was synthesized according to the modified procedure for entry 8 (main text) or entry 9 (SI) in Yan et al. Organometallics 2019. All procedures for carrying out these cis-polymerization were performed in a dry N2 filled glovebox. To a 24 mL vial equipped with a stir bar, 250 mg NHS-monomer 1 (1.06 mmol, 150 equiv.) was added and dissolved in 3-5 mL dry methylene chloride. In a separate 24 mL vial, a solution of 9.7 mg (7.1 μmol, 1 equiv.) catalyst 6 in 2-4 mL dry methylene chloride was prepared. The catalyst formed a bright orange-yellow solution. Note that catalyst 6 typically yielded polymers of greater DP than targeted. To compensate for this, low DP reactions were set up using lower equivalents of monomer to catalyst than theoretically required to yield a polymer of a particular length. Here, a ratio of monomer to catalyst of 150:1 was found to yield approximately DP=200 by 1H NMR end-group analysis. The catalyst solution was added rapidly to the monomer solution and the reaction was allowed to proceed for 30 minutes before removing from the glovebox. The reactant solution rapidly took on a burnt-orange color after initiation. The reactant solution was precipitated dropwise into 40 mL MeOH, centrifuged, and decanted. The product was obtained as a white rock-like solid from precipitation. No Rf was measured for the polymers. DP=200 by 1H NMR end-group analysis where the phenyl signal at 7.44-7.13 ppm was integrated for 5H and the integral of the signal at 1.82 ppm was taken to determine DP. Ð=2.06. Average Yield: 86%. Minimum Yield: 86%. Maximum Yield: 86%. 1H NMR (600 MHz, methylene chloride-d2) δ 7.44-7.13 (m, 5H) 5.58-5.12 (brm, 731H, overlap with solvent), 3.45 (brm, 663H), 2.81 (brm, 1100H), 2.42-1.99 (brm, 440H), 1.82 (brm, 227H), 1.70-1.32 (brm, 163H), 1.25 (brm, 239H).
The silk fibroin polymers were synthesized and characterized as described below. The experiments utilized polymers where glycans were grafted onto the serine residues of boiled silk fibroin. Experiments used polymers at 2.5 wt %.
Silk fibroin (SF) solutions were extracted using our prior protocols. 54 Briefly, 5 grams of Bombyx mori silkworm (Tajima Shoji Co. Ltd., Yokohama, Japan) cut cocoons were degummed in 2 L of 0.02 M Na 2 CO 3 solution (Sigma-Aldrich, St. Louis, MO) in a glass beaker for 60 minutes to remove the sericin protein coating. Degummed silk was collected and rinsed with deionized water (DI) in 4 L (20 minutes, 3 times), followed by drying at room temperature in a fume hood overnight. The dried degummed SF fibers were solubilized in 9.3 M Lithium Bromide (LiBr) (Sigma-Aldrich, St. Louis, MO) solution, in a preheated oven at 60° C. for 4 h. After 4 h, light brown color SF solution was obtained which was dialyzed against 4 L of DI water with six water changes for 48 h (water changes at 1, 2, 4, 24, 36, and 48 h). The dialysis was performed with dialysis tubing (3,500 MWCO, Thermo Scientific, Rockford, IL). After 48 h, the dialyzed silk solution was centrifuged (9,000 RPM, 20 min, 4° C., 2 times) to remove insoluble aggregates. The concentration of the regenerated SF solution was calculated by drying a known mass of the aqueous SF solution in a weigh boat in an oven at 60° C. overnight and assessing the mass of the remaining solid film. The aqueous SF solution was stored at 4° C. until further use.
Aqueous SF solution was carboxylated by nucleophilic substitution reaction in a highly alkaline reaction environment, in the presence of chloroacetic acid (Sigma-Aldrich, St. Louis, MO) at pH ˜13.5. Briefly, the pH of the 1M chloroacetic acid was adjusted by adding freshly prepared 10M sodium hydroxide (NaOH) solution to raise the pH to 13.3-13.5. At pH 13.5, reconstituted SF solution (0.6 wt %) was added dropwise to the mixture. Addition of SF solutions can decrease the pH which was further adjusted to pH 13.5 by dropwise addition of 10 M NaOH solution. The solution was stirred gently for 1 h at RT. After 1 h, sodium phosphate monobasic (NaH 2 PO 4) (Sigma-Aldrich, St. Louis, MO; Lot #015K0024, 4 mg/mL) was added to the reaction mixture and stirred. Addition of NaH 2 PO 4 decreased the pH of the mixture. The pH of the solution was adjusted to 7-7.5 by slow dropwise addition of 10M hydrochloric acid (HCl) (Sigma-Aldrich, St. Louis, MO) solution. At pH 7-7.5, the reaction mixture was stirred for 30 minutes at RT. After 30 minutes, the carboxylated SF solution was dialyzed against DI water for 72 h with six water changes (1 h, 2 h, 4 h, 24 h, 48 h, and 72 h) to remove byproducts and impurities. The dialysis was performed with dialysis tubing (3,500 MWCO, Thermo Scientific, Rockford, IL) in 4 L with gentle stirring. After dialysis, the carboxy-modified SF solutions were filtered in a sterile cell strainer with 40 μm mesh size (Thermo Scientific, Rockford, IL). After filtration, the solutions were frozen at −80° C. overnight followed by lyophilizing for at least 72-96 h. The lyophilized powders were collected and stored at 4° C. until further use.
The carboxylated SF (SF(S)—COOH) was covalently conjugated with primary amines of ethylene diamine (EDA) hydrochloride (Sigma-Aldrich, St. Louis, MO) by carbodiimide coupling in the presence of N-3-Dimethyl amino propyl-N′-ethyl carbodiimide (EDC) hydrochloride, and N-Hydroxy Succinimide (NHS) (Sigma-Aldrich, St. Louis, MO). Briefly, 2 wt % of the SF(S)—COOH solution was dissolved in 0.1M MES (2-(N-morpholino) ethanesulfonic acid) buffer at pH 6. EDA (10×excess) was weighed and pre-dissolved in ultrapure distilled water (Thermo-Fisher Scientific, Waltham, MA) and added to the SF(S)—COOH solution. The pH was readjusted to 6 by dropwise addition of freshly prepared 1M sodium hydroxide (NaOH) solution. EDC (10×) and NHS (10×) (pre-dissolved in MES buffer, pH 6) were added to the reaction mixture at pH 6. The final MES buffer concentration of the reaction mixture was adjusted to 0.05M by addition of ultrapure distilled water. The reaction was stirred gently at RT for 18 h. After the reaction, the aggregates were filtered in a sterile cell strainer with 40 μm mesh size (Thermo Scientific, Rockford, IL) and dialyzed against DI water for at least 72-96 h with six water changes (1, 2, 4, 24, 48, and 72 h). Dialysis was performed with dialysis tubing (3500 MWCO, Thermo Scientific, Rockford, IL). After dialysis, the solutions were frozen at −80° C. overnight followed by lyophilizing for 72 h. The lyophilized powders were stored at 4° C. until further use.
The aminated SF solution (SF(S)-EDA) were conjugated with carboxylic acid moieties of 4-carboxybutyl N-acetyl-β-D-galactosaminide ((3-GalNAc-Bu-COOH) (Sussex Research, Ottawa, Canada), 4-carboxybutyl N-acetyl-β-D-glucosaminide (β-G1cNAc-Bu-COOH) (Sussex Research, Ottawa, Canada), or N-Acetylneuraminic acid hydrate (TCI America, Portland, OR) by carbodiimide coupling in the presence of EDC and NHS (Sigma-Aldrich, St. Louis, MO). Briefly, 2 wt % of the SF(S)-EDA was dissolved in 0.1M MES (2-(N-morpholino) ethanesulfonic acid) buffer at pH 6. β-GalNAc-Bu-COOH (3× times molar excess), β-G1cNAc-Bu-COOH (3× molar excess), or N-Acetylneuraminic acid hydrate (2× molar excess) was weighed and pre-dissolved in 0.1M MES buffer and the pH was readjusted to 6 by dropwise addition of freshly prepared 1M NaOH solution. EDC (3×) and NHS (3×) were added to the sugar solution at pH 6 to activate the carboxylic acid. The reaction was readjusted to pH 6 and stirred for 30 minutes at RT. After 30 minutes, SF(S)-EDA solution was dropwise added to the activated solution. The final MES buffer concentration of the reaction mixture was adjusted to 0.05M by addition of ultrapure water. The pH was readjusted to 6 after addition of SF(S)-EDA solution. The reaction was stirred at RT for 18 h. After 18 h, aggregates were filtered through a sterile cell strainer with 40 μm mesh size (Thermo Scientific, Rockford, IL) and dialyzed against DI water for at least 72 h with six water changes (1 h, 2 h, 4 h, 24 h, 48 h, and 72 h). The dialysis was performed with dialysis tubing (3,500 MWCO, Thermo Scientific, Rockford, IL). After dialysis, the solutions were frozen at −80° C. overnight followed by lyophilizing for 72 h. The lyophilized powders were stored at 4° C. until further use.
SF(S)—COOH was conjugated with amines of D (+)-Glucosamine Hydrochloride (Sigma-Aldrich, St. Louis, MO) by carbodiimide coupling in the presence of EDC and NHS (Sigma-Aldrich, St. Louis, MO) or D (+)-Galactosamine Hydrochloride (Sigma-Aldrich, St. Louis, MO). A 2 wt % of the SF(S)—COOH solution was dissolved in 0.1M MES (2-(N-morpholino) ethanesulfonic acid) buffer at pH 6. D (+)-Glucosamine Hydrochloride (3×) or D(+)-Galactosamine Hydrochloride (3×) was weighed and pre-dissolved in ultrapure distilled water (Thermo-Fisher Scientific, Waltham, MA) and added to the SF(S)—COOH solution. The pH was readjusted to 6 by dropwise addition of freshly prepared 1M sodium hydroxide (NaOH) solution. EDC (3×) and NHS (3×) (pre-dissolved in MES buffer, pH 6) were added to the reaction mixture at pH 6. The final MES buffer concentration of the reaction mixture was adjusted to 0.05M by addition of ultrapure distilled water. The reaction was allowed to stir gently at RT for 18 h. The aggregates were filtered, after 18 h, through a sterile cell strainer (40 μm mesh size) (Thermo Scientific, Rockford, IL) and dialyzed against DI water for at least 72 hours with six water changes (1, 2, 4, 24, 48, and 72 h). Dialysis was performed with dialysis tubing (3500 MWCO, Thermo Scientific, Rockford, IL). After dialysis, the solutions were frozen at −80° C. overnight followed by lyophilizing for 72 h. The lyophilized powders were stored at 4° C. until further use.
The regenerated silk fibroin was covalently conjugated with primary amines of ethylene diamine (EDA) hydrochloride (Sigma-Aldrich, St. Louis, MO) by carbodiimide coupling in the presence of EDC, and NHS (Sigma-Aldrich, St. Louis, MO). Briefly, 2 wt % of the SF solution was dissolved in 0.1M MES (2-(N-morpholino) ethanesulfonic acid) buffer at pH 6. EDA (10×) was weighed and pre-dissolved in Ultrapure distilled water (Thermo-Fisher Scientific, Waltham, MA) and added to the aqueous SF solution. The pH was readjusted to 6 by dropwise addition of freshly prepared 1M sodium hydroxide (NaOH) solution. EDC (10×) and NHS (10×) (pre-dissolved in MES buffer, pH 6) were added to the reaction mixture at pH 6. The final MES buffer concentration of the reaction mixture was adjusted to 0.05M by addition of ultrapure distilled water. The reaction was stirred gently at RT for 18 h. After the reaction was completed, aggregates were filtered through a cell strainer (mesh size 40 μm) (Thermo Scientific, Rockford, IL) and dialyzed against DI water for at least 72 hours with six water changes (1, 2, 4, 24, 48, and 72 h). Dialysis was performed with dialysis tubing (3500 MWCO, Thermo Scientific, Rockford, IL). After dialysis, the solutions were frozen at −80° C. overnight followed by lyophilizing for 72 h. The lyophilized powders were stored at 4° C. until further use.
The aminated SF solution (SF (D, E)-EDA) were conjugated with carboxylic acid moieties of 4-carboxybutyl N-acetyl-β-D-galactosaminide (β-GalNAc-Bu-COOH) (Sussex Research, Ottawa, Canada) by carbodiimide coupling in presence of EDC and NHS (Sigma-Aldrich, St. Louis, MO). Briefly, 2 wt % of the SF (D, E)-EDA was dissolved in 0.1M MES (2-(N-morpholino) ethanesulfonic acid) buffer at pH 6. β-GalNAc-Bu-COOH (3×times molar excess) was weighed and pre-dissolved in 0.1M MES buffer and pH was readjusted to 6 by dropwise addition of freshly prepared 1M NaOH solution. EDC (3×) and NHS (3×) were added to β-GalNAc-Bu-COOH at pH 6 to activate the carboxylic acid. The reaction was readjusted to pH 6 and stirred for 30 minutes at RT. After 30 minutes, the SF (D, E)-EDA solution was added dropwise to the activated solution. The final MES buffer concentration of the reaction mixture was adjusted to 0.05M by addition of ultrapure water. The pH was readjusted to 6 after addition of SF (D, E)-EDA solution. The reaction was stirred at RT for 18 h. After the reaction was complete, aggregates were filtered through a sterile cell strainer with 40 μm mesh size (Thermo Scientific, Rockford, IL) and dialyzed against DI water for at least 72 h with six water changes (1 h, 2 h, 4 h, 24 h, 48 h, and 72 h). Dialysis was performed with dialysis tubing (3,500 MWCO, Thermo Scientific, Rockford, IL). After dialysis, the solutions were frozen at −80° C. overnight followed by lyophilizing for 72 h. The lyophilized powders were stored at 4° C. until further use.
MUC5B Mucin Purification from Pooled Saliva
Submandibular saliva was collected from informed consenting volunteers as described previously.40 Saliva donations from five donors were pooled before MUC5B was purified using liquid chromatography as described previously.55 Purified MUC5B was flash cooled, lyophilized, and stored at −80° C. Before use, MUC5B was rehydrated in Milli-Q-purified water and agitated at 4° C. overnight. Protocols involving samples from human participants were approved by the Massachusetts Institute of Technology's Committee on the Use of Humans as Experimental Subjects.
Gardnerella vaginalis 14018 ATCC strain was obtained from ATCC. Salivary MUC5B mucin was isolated and purified as described previously. Galactose and D-Fucose were obtained from Sigma-Aldrich. AnaeroPacks were purchased from VWR. Prior to the experiment, MUC5B mucin was weighed out and solubilized to desired concentration on a shaker overnight.
To establish mature biofilms, an overnight preculture of Gardnerella vaginalis ATCC 14018 was diluted 1:20 into fresh NYC III medium with 1% glucose and incubated at 37° C. in the anaerobic chamber for 24 h.
Before adding fresh solutions, a set of triplicate wells was sacrificed to quantify cell counts in supernatant and biofilm fraction at the 24 h timepoint. The spent supernatant was gently drained from the rest of the wells and fresh medium or mucin/monosaccharide solutions in medium were added on top of the bacterial biomass.
Plate was gently rocked (100 rpm) for 24 h in the anaerobic atmosphere (generated with AnaeroPack, Mitsubishi in the airtight container) in the 37° C. incubator. After 24 h, the supernatant was removed into a separate plate and the biofilms were washed twice with 1004, sterile phosphate-buffer saline (PBS) and washes were combined with the supernatant (=supernatant fraction). Another 1004, of PBS was added to the biofilms, each well was vigorously scraped with P20 tip for 20 s and scraped biomass pipetted up and down to homogenize the solution (=biofilm fraction). Both supernatant and biofilm solutions were serially diluted, plated on BHI-agar (BHI=brain-heart infusion, 1.5% agar) and incubated in the anaerobic chamber 37° C. incubator. Colonies were counted after ˜48 h.
Metronidazole was purchased from Combi-Blocks. Galactose and D-Fucose were obtained from Sigma-Aldrich.
Mature biofilms of Gardnerella vaginalis were established as described above. On a separate plate serial dilution of metronidazole alone or in combination with 0.2 wt % Gal/D-Fuc were prepared in NYC III 1% Glc medium. Negative controls (no metronidazole) were also included on the plate. After spent supernatant was drained from mature biofilms, 100 μL, of metronidazole/metronidazole-monosaccharide or negative controls solutions were added to the biofilms and plate was rocked (100 rpm) for 24 h in the anaerobic atmosphere (generated with AnaeroPack, Mitsubishi in the airtight container) in the 37° C. incubator. After 24 h incubation, the CFUs of supernatant and biofilm fractions were quantified as described in the biofilm disruption experiment.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.
It should be understood that the subject matter defined in the appended claims is not necessarily limited to the specific implementations described above.
This application claims the benefit of U.S. Provisional Application No. 63/351,552, filed on Jun. 13, 2022. The entire teachings of the above application are incorporated herein by reference.
This invention was made with government support under EB017755 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63351552 | Jun 2022 | US |
