WO2018057747A1

WO2018057747A1 - Pharmaceutical compositions and methods for delivering microbial compositions

Info

Publication number: WO2018057747A1
Application number: PCT/US2017/052734
Authority: WO
Inventors: Eva DE LA SERNA; Dean Liang GLETTIG
Original assignee: Finch Therapeutics, Inc.
Priority date: 2016-09-21
Filing date: 2017-09-21
Publication date: 2018-03-29

Abstract

The present invention relates to, in part to various pharmaceutical compositions, methods for delivering a microbial composition to the gastrointestinal tract of a subject, methods for forming a delayed-release capsule, and methods for forming a capsule filled with microbial composition in an aqueous phase.

Description

PHARMACEUTICAL COMPOSITIONS AND METHODS FOR DELIVERING MICROBIAL

COMPOSITIONS

FIELD OF THE INVENTION

This invention generally relates to articles (e.g., capsules) and methods for delivering microbial compositions, such as for the administration of microbial compositions to a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to US Provisional Patent Application No. 62/397,870, filed September 21 , 2016 and to US Provisional Patent Application No 62/397,872, also filed September 21 , 2016. The entire contents of the aforementioned patent applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Fecal microbiota transplantation (FMT) is known to be a highly effective treatment for recurrent C. difficile infections and holds significant promise for a number of diseases associated with dysbiosis of the microbiome. Current efforts in this field are focused primarily on the use of human stool, however future therapies may instead rely on synthetic cocktails of bacteria isolated from the human Gl tract or other environments and grown in pure or mixed cultures. Both for natural and synthetic products, effective techniques are needed to stabilize material during storage and facilitate delivery to the appropriate site within the Gl tract. Material stabilization has typically been accomplished through cryopreservation with glycerol and long-term storage in ultra-low temperature freezers (e.g., -80 °C). Traditionally, material has been delivered either through the lower Gl tract via colonoscopy, sigmoidoscopy or enema or to the upper Gl tract via naso-gastric, naso-duodenal or naso-jejunal intubation. Colonoscopy and sigmoidoscopy are highly effective, but both carry significant procedure related costs and risks. Enema delivery is inexpensive, less invasive and has fewer procedure related risks, but it is less effective empirically, partly because of challenges in material retention and because less material is delivered to the upper large intestine. Enema delivery is also unfavorable to many patients. Traditional approaches to upper-GI administration carry risks of aspiration related complications (particularly naso-gastric delivery) and are invasive, uncomfortable procedures that carry moderate costs. Oral administration alternatives generally also fail to provide targeted delivery to the diseased site of the patient. Accordingly, improved articles and methods are needed. SUMMARY OF THE INVENTION

The present invention generally relates to articles and methods comprising microbial compositions, such as for the administration of microbial compositions to a subject.

One aspect of the present invention is a pharmaceutical composition for administration of a microbial composition in an aqueous phase. The pharmaceutical composition comprises a capsule including a capsule body and a capsule cap comprising a degradable material, the capsule having an inner surface and an outer surface, wherein an inner layer comprising a hydrophobic material coats the inner surface and an outer layer comprising a delayed release material coats the outer surface; and an aqueous phase comprising the microbial composition encapsulated by the capsule. The inner layer inhibits or substantially reduces dissolution of the capsule.

In embodiments, the degradable material at least partially degrades or dissolves in the presence of an aqueous phase at ambient conditions.

In embodiments, the degradable material is selected from the group consisting of gelatin and a derivative thereof, HPMC, alginate and a derivative thereof, polylactic acid, polyglycolic acid, copolymers thereof, and a combination thereof. In embodiments, the degradable material is gelatin.

In embodiments, a portion of the capsule would degrade, dissolve, leak, and/or crack if contacted with the aqueous phase. In embodiments, the portion of the capsule would degrade, dissolve, leak, and/or crack less than 1 day, 12 hours, 6 hours, 3 hours, or 1 hour after contacting the aqueous phase.

In embodiments, the capsule further comprises an enteric polymer, e.g., the enteric polymer comprises a EUDRAGIT® (poly(meth)acrylate).

In embodiments, the inner layer is disposed between the aqueous phase and the inner surface of the capsule.

In embodiments, the inner layer covers substantially the entire inner surface of the capsule body, e.g., the inner layer covers the entire inner surface of the capsule body.

In embodiments, the inner layer does not cover a portion of the inner surface of the capsule cap, e.g., the portion of the inner surface of the capsule cap that is not covered by the inner layer is located at the open end of the cap. In embodiments, the portion of the inner surface of the capsule cap that is not covered by the inner layer includes the portion of the capsule cap that overlaps the capsule body when the capsule is sealed.

In embodiments, hydrophobic material of the inner layer is selected from the group consisting of zein, polysaccharides, silk, polycaprolactone, oil, pectin, wax, polymers, shellac and a derivative thereof, and a combination thereof. In embodiments, the hydrophobic material of the inner layer is shellac. In embodiments, the inner layer further comprises an enteric polymer, e.g., the enteric polymer comprises a EUDRAGIT® (poly(meth)acrylate).

In embodiments, the inner layer prevents or inhibits the aqueous phase from contacting the degradable material of the capsule. In embodiments, the inner layer prevents or inhibits the aqueous phase from degrading, dissolving, and/or cracking the capsule and/or causing the capsule to leak. In embodiments, a portion of the capsule lacking an inner layer would degrade, dissolve, leak, and/or crack less than 1 day, 12 hours, 6 hours, 3 hours, or 1 hour after contacting the aqueous phase.

In embodiments, the outer layer covers substantially the entire outer surface of the capsule, e.g., the outer layer covers the entire outer surface of the capsule.

In embodiments, the delayed release material comprises an enteric polymer, e.g., the enteric polymer comprises a EUDRAGIT® (poly(meth)acrylate) and the enteric polymer comprises Phloral®.

In embodiments, the outer layer prevents, inhibits, or delays a moist or aqueous external environment from contacting the degradable material of the capsule. In embodiments, the outer layer prevents, inhibits, or delays the moist or aqueous external environment from degrading, dissolving, and/or cracking the capsule and/or causing the capsule to leak. In embodiments, a portion of the capsule lacking an outer layer would degrade, dissolve, leak, and/or crack less than 12 hours, 6 hours, 4 hours, 2 hours, or 1 hour after contacting the moist or aqueous external environment.

In embodiments, the pharmaceutical composition is stable at room temperature for at least 1 day, 7 days, 1 month, or 1 year. In embodiments, the pharmaceutical composition is stable at 37 °C at a pH less than 7, pH less than 6, a pH less than 5 a pH less than 4, a pH less than 3, or a pH less than 2 for at least 1 hour. In embodiments, the pharmaceutical composition begins to dissolve, degrade, crack, and/or leak in less than 6 hours, 4 hours, 2 hours, or 1 hour at 37 °C at a pH less greater than 7.

In embodiments, pharmaceutical composition begins to dissolve, degrade, crack, and/or leak in less than 4 hours, 2 hours, at 1 hour at 37 °C at a pH less greater than 7. In embodiments, the pharmaceutical composition begins to dissolve, degrade, crack, and/or leak in less than 6 hours, 4 hours, 2 hours, or 1 hour at 37 °C at a pH less greater than 8.

In embodiments, the microbial composition is human stool or a derivative thereof.

In embodiments, the microbial composition comprises bacteria, fungi, phages, viruses, fiber, and/or mucus, e.g., the microbial composition comprises viable bacteria.

In embodiments, the capsule, the inner layer, the outer layer, and/or the aqueous phase comprises an additive, e.g., an additive is selected from the group consisting of starches, genipin, aloe, whey protein, guar gum, xantham gum, and carrageenan.

Another aspect of the present invention is a method for delivering a microbial composition to the gastrointestinal (Gl) tract of a subject. The method comprises a step of administering a pharmaceutical composition of any of the above claims to the subject. In embodiments, the Gl tract comprises one or more of the duodenum, small intestine, duodenum, jejunum, ileum, large intestine, colon transversum, colon descendens, colon ascendens, colon sigmoidenum, cecum, and rectum. In embodiments, the administering is oral administration. In embodiments, the subject has a disease/disorder associated with the presence of abnormal enteric microflora.

Yet another aspect of the present in is a method for forming a delayed-release capsule. The method comprises steps of forming an outer layer which coats at least a portion of an outer surface of a capsule body and forming an outer layer which coats at least a portion of the outer surface of a capsule cap with a solution comprising a delayed release material; and forming an inner layer which coats at least a portion of an inner surface of a capsule body and forming an inner layer which coats at least a portion of the inner surface of a capsule cap with a solution comprising a hydrophobic material.

In embodiments, the step of forming an outer layer precedes the step of forming an inner layer. In embodiments, the solution comprising a delayed release material is prepared by dispersing one or more enteric polymers in a solution.

In embodiments, the step of forming an outer layer comprises spraying the solution comprising a delayed release material onto the outer surface of a capsule body and/or onto the outer surface of a capsule cap.

In embodiments, the outer layer covers substantially the entire outer surface of the capsule body and the capsule cap, e.g., the outer layer covers the entire outer surface of the capsule body and the capsule cap.

In embodiments, the enteric polymer comprises a EUDRAGIT® (poly(meth)acrylate). In embodiments, the solution comprises a delayed release material comprises at least 50% EUDRAGIT® (poly(meth)acrylate). In embodiments, the enteric polymer comprises Phloral®.

In embodiments, the outer layer is dried for between about 8 hours and about 24 hours.

In embodiments, the solution comprising a hydrophobic material is prepared by dispersing one or more hydrophobic materials in a solution.

In embodiments, the solution comprising a hydrophobic material comprises an alcohol, e.g., ethanol, e.g., a 200-proof ethanol.

In embodiments, the hydrophobic material is selected from the group consisting of zein, polysaccharides, silk, polycaprolactone, oil, pectin, wax, polymers, shellac and a derivative thereof, and a combination thereof. In embodiments, the hydrophobic material is shellac.

In embodiments, the solution comprising a hydrophobic material comprises between about 0.1 g shellac per ml ethanol to about 1 .3 g shellac per ml of ethanol, e.g., between about 0.7, 0.8, 0.9, and 1 .0 g shellac per ml ethanol. In embodiments, the solution comprising a hydrophobic material comprises a 1 :1 ratio of shellac (in grams) to ml of ethanol.

In embodiments, the step of forming an inner layer on at least a portion of an inner surface of a capsule body comprises substantially filling the capsule body with the solution comprising a hydrophobic material, removing the solution comprising the hydrophobic material, and drying the capsule body for about 24 hours to about three days.

In embodiments, the step of forming an inner layer on at least a portion of an inner surface of a capsule cap comprises filling the capsule cap up to a defined level of the capsule cap with the solution comprising a hydrophobic material, removing the solution comprising a hydrophobic material, and drying the capsule cap for 24 hours to three days. In embodiments, the defined level of the capsule cap excludes the portion of the capsule cap that overlaps the capsule body when the capsule is sealed. In embodiments when the capsule is a size 00 capsule, between about 50 μΙ and about 400 μΙ of the hydrophobic coating solution of the hydrophobic coating solution is dispensed per capsule cap.

In embodiments, when the capsule is a size 00 capsule, a capsule body comprising an inner layer weighs between about 0.4 g and about 0.9 g and a capsule cap comprising an inner layer weighs between about 0.01 g and about 0.03 g; depending on which components are included in an inner layer solution and/or in an outer layer solution, the weight of a coated capsule body and coated a capsule cap may be higher than the specified weights or may be lower than the specified weights.

In embodiments, the capsule comprises a degradable material that is selected from the group consisting of gelatin and a derivative thereof, HPMC, alginate and a derivative thereof, polylactic acid, polyglycolic acid, copolymers thereof, and a combination thereof. In embodiments, the degradable material is gelatin.

In another aspect, the present invention provides a method for forming a capsule filled with microbial composition in an aqueous phase. The method comprises steps of obtaining the delayed- release capsule obtained by the method of any above aspect or embodiment, filling the capsule body with the microbial composition in an aqueous phase, and sealing the capsule body with a capsule cap. In embodiments, the microbial composition is human stool or a derivative thereof. In embodiments, the microbial composition comprises bacteria, fungi, phages, viruses, fiber, and/or mucus, e.g., the microbial composition comprises viable bacteria.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein. Embodiments of the invention will now be described with reference to the Drawings and following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

FIG. 1 shows an illustrative embodiment of an article for delivering a microbial composition, according to one set of embodiments.

FIG. 2 shows an illustrative embodiment of an article for delivering a microbial composition, according to one set of embodiments.

FIG. 3A to FIG. 3C show illustrate embodiments of a capsule precursor and methods for coating the capsule precursor, according to one set of embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Articles and methods comprising microbial compositions, such as articles for the administration of microbial compositions to a subject, are generally provided. The articles and methods may include a capsule. In some embodiments, the capsule is coated internally such that an aqueous phase encapsulated by the capsule does not rapidly (e.g., in less than 24 hours at room temperature) degrade the capsule and/or the capsule materials. The capsule may be designed to have desirable properties such as stability against degradation at room temperature and/or 37 °C for relatively long times. For example, the capsule may not leak, crack, or otherwise be dissolved by the encapsulated aqueous phase for at least 1 day at room temperature, or at least 1 hour at 37 °C. In some embodiments, the capsule encapsulates viable microbial compositions. The capsule may be designed, in some cases, to be administered to a subject such that the microbial composition is released from the capsule at a location internally to the subject. Such capsules may be useful for the administration (e.g., oral administration) of microbial compositions to a subject for fecal matter transplantation (FMT)-based therapies and/or for treatment of selected diseases including, but not limited to, C. difficile infections.

In an exemplary embodiment, the article comprises a capsule comprising a degradable material, an aqueous phase comprising a microbial composition encapsulated by the capsule, and an inner layer coated internally in the capsule and disposed between the aqueous phase and the degradable material. In some embodiments, the capsule is coated externally with an outer layer (e.g., comprising an enteric polymer). In embodiments, the capsule is dual-coated, i.e., coated internally with an inner layer and coated externally with an outer layer. In some embodiments, the capsule is stable at room temperature under ambient conditions for at least 1 day. In certain embodiments, the microbial composition comprises one or more viable bacteria. In some embodiments, the bacteria are not lyophilized.

Advantageously, the articles and methods described herein may permit the administration (e.g., oral administration) and release of viable microbial compositions to selected locations internally to a subject, without the need for invasive and often expensive procedures such as colonoscopy, sigmoidoscopy, enema, and/or naso-gastric, naso-duodenal or naso-jejunal intubation. In some embodiments, the microbial compositions are retained in an aqueous phase, reducing the loss of viability that is generally intrinsic to dewatering-based methods of preservation such as lyophilization or other dewatering techniques. In certain embodiments, the aqueous phase of the microbial composition does not rapidly or substantially degrade and/or dissolve the encapsulating material (e.g., the capsule) via the incorporation of an inner layer to the capsule, permitting stable delivery of the microbial composition in devices comprising materials such as gelatin-based capsules, HPMC-based capsules, polysaccharide microcapsules, or in lipid-based microcapsules.

Advantageously, the articles described herein may permit the storage of encapsulated microbial compositions at low temperatures (e.g., -80 °C) without damaging the physical integrity and strength of the capsule. As described further below, in some embodiments, the capsule is coated with an outer layer that degrades at a particular pH (e.g., an enteric polymer that at least partially degrades in the pH of the human colon) for targeted delivery of the microbial compositions. In certain embodiments, the outer layer undergoes enzymatic degradation (e.g., comprising a polymer that undergoes targeting degradation by an enzyme present in a location internally to a subject). In alternative embodiments, the capsule does not comprise an outer layer.

Capsule

The capsule may comprise any suitable material. Those skilled in the art would understand based upon the teachings of this specification that the term capsule is not intended to be limited to the types of capsules described in, for example, the United States Pharmacopeia (USP), although they may comprise such capsules, but refers to any container or layer which encloses and/or encapsulates a material and/or liquid such as the aqueous phase comprising microbial compositions described herein. In certain embodiments, the capsule (and/or capsule material) is designed and selected to provide mechanical stability to the article. For example, in some embodiments, the capsule maintains a particular shape (e.g., a cylinder with hemispherical ends) such that it encapsulates an aqueous phase (e.g., an aqueous phase comprising a microbial composition) without leaking of the aqueous phase from the article.

In certain embodiments, the capsule has a particular shape or size. For example, in some cases, the capsule has a shape or size as described in the USP including, but not limited to, 000 capsule, 00E capsule, 00 capsule, 0E capsule, 0 capsule, 1 capsule, 2 capsule, 3 capsule, 4 capsule, or 5 capsule. In some embodiments, the capsule comprises a particle (e.g., a spherical particle, a microparticle). Other capsule shapes and/or sizes are also possible.

In some embodiments, the capsule comprises a polymeric material. Non-limiting examples of suitable polymeric materials include gelatin, polymethylmethacrylate, poly(N,N-dimethylacrylamide), polyoxamer, polyethylene glycol, polypropylene glycol, polysaccharides (e.g., sucrose, trehalose, glucose, starches such as tapioca and arrowroot, chitosan, alginate, guar gum), polyacrylate, polymethacrylate, polyvinyl alcohol, polyalkylene glycols, polyacrylamide, polyvinylpyrrolidone, polyurethane, polylactide, lactide/glycolide copolymer, polycaprolactone, polydioxanones, polyanhydride, polyhydroxybutyrate, polysiloxane, polytrimethylene carbonate, polyalkylene glycol, and combinations and/or copolymers thereof. In embodiments, the capsule comprises gelatin.

In some embodiments, the capsule comprises a degradable material. As used herein, a degradable material refers to a material capable of undergoing the breakdown of covalent bonds in the present of particular conditions. In some embodiments, the degradable material at least partially degrades in the presence of water at ambient conditions in less than 1 hour. For example, a capsule comprises a degradable material that at least partially degrades in the presence of water at ambient conditions in less than 1 hour, would leak any liquid contents contained therein in less than the 1 hour. In certain embodiments, the degradable material is biodegradable (e.g., breaks down when to physiological conditions). In some embodiments, the degradable material breaks down at a particular pH.

A screening test may be used to determine suitable degradable materials for the capsule. For example, the capsule material may be selected from materials that encapsulate water absent an inner layer disposed between the material and the aqueous phase for less than 1 hour at room temperature and under ambient conditions. That is to say, within 1 hour, the water would dissolve and/or degrade the capsule material such that the water leaks from the capsule. In some embodiments, the degradable material is selected from the group consisting of gelatin and derivatives thereof, HPMC, alginate and derivatives thereof, polylactic acid, polyglycolic acid, copolymers thereof, and combinations thereof.

In certain embodiments, the capsule may comprise a bioadherent polymer such as mucin. In certain embodiments, the capsule comprises an enteric polymer. In some embodiments, the enteric polymer is selected such that the capsule is stable in an acidic gastric environment [i.e., having a pH1 to pH4) but dissolves in a more alkaline region of the gastrointestinal tract [i.e., having a pH greater than 5.5). In some embodiments, the enteric polymer includes, but is not limited to, cellulose acetate phthalate (CAP), hypromellose (INN) hydroxypropyl methylcellulose (HPMC), poly(methacrylic acid-co- ethyl acrylate) (e.g., EUDRAGIT®, available from Evonik Industries AG (Essen, Germany)), derivatives thereof, and copolymers thereof.

In some embodiments, the capsule comprises a material that dissolves and/or degrades at a particular pH after a given amount of time (e.g., after at least an hour of exposure to the particular pH). In some embodiments, the capsule is designed to at least partially degrade and/or at least partially dissolve in the colon of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 7.0 (e.g., EUDRAGIT® S 100, EUDRAGIT® S 12, 5, EUDRAGIT® FS 30D, Phloral®); see, e.g., US 9,023,368, US 20150150837, and US 20150202162. In certain embodiments, the capsule is designed to at least partially dissolve and/or at least partially degrade in the duodenum of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 5.5 (e.g., EUDRAGIT® L 30D-55, EUDRAGIT® L 100-55). In some embodiments, the capsule is designed to at least partially dissolve and/or at least partially degrade in the jejunum of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 6.0 and less than or equal to 7.0 (e.g., EUDRAGIT® L 100, EUDRAGIT® L 12, 5). In certain embodiments, the capsule is designed to at least partially dissolve and/or at least partially degrade in the stomach of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 1 .0 and less than or equal to 5.0 (e.g., EUDRAGIT® E 100, EUDRAGIT® E 12, 5, EUDRAGIT® E PO).

In some embodiments, the enteric polymer is selected such that it exhibits time controlled degradation and/or dissolution, independent of pH. Non-limiting examples of such enteric polymers include EUDRAGIT® RL 30D, EUDRAGIT® RL PO, EUDRAGIT® RL 100, EUDRAGIT® RL 12, 5, EUDRAGIT® RS 30D, EUDRAGIT® RS PO, EUDRAGIT® RS 100, EUDRAGIT® RS 12, 5, EUDRAGIT® NE 30D, EUDRAGIT® NE 40D, and EUDRAGIT® NM 30D. In certain embodiments, the capsule comprises a copolymer of two or more enteric polymers described herein.

In some embodiments, the capsule comprises an enteric elastomer. In some embodiments, the enteric elastomer comprises a mixture of two or more polymers with carboxyl functionality such that the two or more polymers form hydrogen bonds with one another and has both enteric and elastic properties. In certain embodiments, the enteric elastomer comprises a first polymer comprising a structure as in Formula (I):

or a pharmaceutically acceptable salt thereof, wherein each R¹ is the same or different and is selected from the group consisting of optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted arylene, and optionally substituted heteroarylene, each R² is the same or different and is selected from the group consisting of hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyi, each R³ is the same or different and is selected from the group consisting of optionally substituted alkylene and optionally substituted heteroalkylene, n is an integer between 25 and 250,000, and a second polymer comprising a structure as in Formula (II) hydrogen bonded to the first polymer:

or a pharmaceutically acceptable salt thereof, wherein each R⁴ is the same or different and is selected from the group consisting of optionally substituted alkylene and optionally substituted heteroalkylene, each R⁵ is the same or different and is selected from the group consisting of optionally substituted alkylene and optionally substituted heteroalkylene, each R⁶ is the same or different and is selected from the group consisting of hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyi, each R⁷ is the same or different and is selected from the group consisting of hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyi, each R8 is the same or different and is optionally substituted alkyl, p is an integer between 1 and 10, q is an integer between 1 and 10, and z is an integer between 1 and 150,000, provided that (p + q)*z is greater than or equal to 20. Suitable enteric elastomers and methods for making such enteric elastomers are described in more detail in International Patent Publication No. WO 2015/191922, entitled "Enteric Elastomers," published on December 17, 2015, and filed on June 1 1 , 2015 as International Patent Application Serial No. PCT/US 15/35425, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, the capsule comprises a polymer formed by the reaction of one or more monomers in the presence of a food grade catalyst (e.g., caffeine). Suitable polymers formed in the presence of food grade catalysts are described in more detail in International Patent Publication No. WO 2015/168297, entitled "Polymeric Materials for Bio-Applications," published on November 5, 2015, and filed on April 29, 2015 as International Patent Application Serial No. PCT/US15/28311 , which is incorporated herein by reference in its entirety for all purposes.

Drug Product

As described herein, in some embodiments, the capsule comprises a drug product in an aqueous phase. In some embodiments, the aqueous phase comprises water. In embodiments, the aqueous phase comprises glycerol. In certain embodiments, the drug product comprises a microbial composition. The microbial composition may be stool or derivatives thereof. In an exemplary embodiment, the microbial composition is human stool or derivatives thereof. In some embodiments, the microbial composition comprises one or more of bacteria, fungi, phages, viruses, fiber, and/or mucus. In some such embodiments, the bacteria, fungi, phages, and/or viruses may be isolated. For example, bacteria (and/or fungi, phases, viruses) may be isolated from a first subject (e.g., a first human subject), encapsulated in the articles described herein, and administered to a second subject (e.g., a second human subject) as described herein. In some embodiments, the bacteria comprise aerobic bacteria. In certain embodiments, the bacteria comprise anaerobic bacteria.

In embodiments in which the microbial composition comprises bacteria, the bacteria may be viable. The term "viable" as used herein generally refers to the ability of an organism, such as bacteria, to survive under particular conditions. In some embodiments, the capsule comprises bacteria which remains viable for greater than or equal to 1 day, greater than or equal to 2 days, greater than or equal to 3 days, greater than or equal to 7 days, greater than or equal to 14 days, greater than or equal to 30 days, greater than or equal to 90 days, or greater than or equal to 180 days at room temperature under ambient conditions.

In embodiments the term "viable" refers to the ability of an organism, such as bacteria, to survive, to persist, to proliferate, and/or to have metabolic activity when contained inside a capsule of the present invention and/or once released from the capsule of the present invention.

In embodiments the term "viable" refers to the ability of an organism, such as bacteria, to survive within the gastrointestinal environment, to persist within the gastrointestinal environment, to proliferate within the gastrointestinal environment, to have metabolic activity upon introduction into a gastrointestinal environment, and/or to engraft within the gastrointestinal environment.

With respect to a spore-forming bacterium, the term "viable" further includes an ability of the spore-forming bacterium in a microbial composition to form spores, e.g., spores that are able to germinate within the gastrointestinal environment.

In certain embodiments, features of a capsule of the present invention, e.g., the presence of the inner layer, protects the capsule from the aqueous phase for less than or equal to 365 days, less than or equal to 180 days, less than or equal to 90 days, less than or equal to 30 days, less than or equal to 14 days, less than or equal to 7 days, less than or equal to 3 days, or less than or equal to 2 days at room temperature, e.g., under ambient conditions. Combinations of the above-referenced ranges are possible (e.g., greater than or equal to 1 day and less than or equal to 365 days).

Those skilled in the art would understand based upon the teaching of this specification that the viability of the bacteria encapsulated and/or administered in the articles described herein may be significantly greater than the viability of an equal amount of the same bacteria that has been lyophilized. In some embodiments, the microbial composition comprises bacteria which has not been and/or is not lyophilized.

In some embodiments, the drug product may comprise a therapeutic agent in addition to a microbial composition (as described herein). As used herein, the term "therapeutic agent", also referred to as a "drug", refers to an agent that is administered to a subject to treat a disease, disorder, or other clinically recognized condition, or for prophylactic purposes, and has a clinically significant effect on the body of the subject to treat and/or prevent the disease, disorder, or condition. Therapeutic agents include, without limitation, agents listed in the United States Pharmacopeia (USP), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Ed., McGraw Hill, 2001 ; Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange; 8th edition (September 21 , 2000); Physician's Desk Reference (Thomson Publishing), and/or The Merck Manual of Diagnosis and Therapy, 17th ed. (1999), or the 18th ed (2006) following its publication, Mark H. Beers and Robert Berkow (eds.), Merck Publishing Group, or, in the case of animals, The Merck Veterinary Manual, 9th ed., Kahn, C.A. (ed.), Merck Publishing Group, 2005. In some embodiments, the therapeutic agent may be selected from "Approved Drug Products with Therapeutic Equivalence and Evaluations," published by the United States Food and Drug Administration (F.D.A.) (the "Orange Book"). In some cases, the therapeutic agent is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body. For example, drugs approved for human use are listed by the FDA under 21 C.F.R. §§ 330.5, 331 through 361 , and 440 through 460, incorporated herein by reference; drugs for veterinary use are listed by the FDA under 21 C.F.R. §§ 500 through 589, incorporated herein by reference. All listed drugs are considered acceptable for use in accordance with the present invention. In certain embodiments, the therapeutic agent is a small molecule. Exemplary classes of agents include, but are not limited to, analgesics, anti-analgesics, antiinflammatory drugs, antipyretics, antidepressants, antiepileptics, antipsychotic agents, neuroprotective agents, anti-proliferatives, such as anti-cancer agents (e.g., taxanes, such as paclitaxel and docetaxel; cisplatin, doxorubicin, methotrexate.), antihistamines, antimigraine drugs, hormones, prostaglandins, antimicrobials (including antibiotics, antifungals, antivirals, antiparasitics), antimalarials, antimuscarinics, anxioltyics, bacteriostatics, immunosuppressant agents, sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthma drugs, cardiovascular drugs, anesthetics, anti-coagulants, inhibitors of an enzyme, steroidal agents, steroidal or non-steroidal anti-inflammatory agents, immunosuppressive agents, corticosteroids, dopaminergics, electrolytes, gastro-intestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, anorectics, anti- narcoleptics, and nutraceuticals such as vitamins, supplements such as calcium or biotin, or natural ingredients such as plant extracts or phytohormones.

Methods for preparing a drug product are described elsewhere, e.g., EP2951283, EP2951285, EP2953472, EP2953474, EP2956006, EP2967077, US20120149584, US20140363397, US20140363398, US20140363400, US20150216917, US20150238544, US20150238546, US20150297642, US20160030494, US20160151432, US20160151433, US20160158294, US20160158295, US20160243172, US20160296568, US20160331791 , US20160354414, US20160361263, US20160367607, US20160375067, US20160375068, US20170020932, US20170027996, US20170049826, US20170049827, US6645530, US8460648, US8906668, US9011834, US9040036, US9050358, US9180147, US9408872, US9433651 , US9446080, US9463208, US9468658, US9511099, US9511 100, US9533014, US9572842, US9610307, US9623056, US9629881 , US9642880, WO2011033310, WO201 1046616, WO201 1094027, WO2011 152566, WO2012016287, WO2012122478, WO2013053836, WO2013080561 , WO2014078911 , WO2014152484, WO2014176632, WO2015006355, WO2016065279, WO2016070151 , WO2016086205, WO2016183577, WO2016201114, and WO2017008026, the contents of which are incorporated herein by reference in their entirety.

Inner Layer

In some embodiments, the article comprises an inner layer. In certain embodiments, the inner layer separates an aqueous phase from the capsule, thereby inhibiting dissolution of a capsule and/or promoting stability of the capsule. In embodiments, the article comprises two or more inner layers which are sequentially applied, e.g., after a first inner layer has been applied and air dried, a second inner layer is applied. Unless the context clearly dictates otherwise, as used herein, the term "inner layer" includes at least one inner layer, e.g., one, two, three, four, or more inner layers. In embodiments, a capsule cap or a capsule body is provided as many layers of hydrophobic coating until a desired weight range is achieved, e.g., bodies about 0.3 g to 1 .2 g and caps: about 0.005 g to about 0.06 g.

FIG. 1 is an illustrative embodiment of an article (e.g., for administration of a microbial composition) including a capsule as described herein. In some embodiments, an article 100 comprises an aqueous phase 1 10 encapsulated by a capsule 130. In certain embodiments, an inner layer 120 is adjacent to capsule 130 and disposed between capsule 130 and aqueous phase 110. Inner layer 120 may be coated directly onto [i.e., directly adjacent) at least an inner surface of capsule 130. As used herein, when a layer is referred to as being "adjacent" another layer, it can be directly adjacent to the layer, or an intervening layer also may be present. A layer that is "directly adjacent" to another layer means that no intervening layer is present..

As described herein, the inner layer may be selected and designed such that it protects the capsule (or capsule material) from the aqueous phase. For example, in some embodiments, the inner layer protects the capsule from the aqueous phase, e.g., helps prevent the aqueous phase from contacting the capsule and/or inhibits or substantially reduces dissolution, degradation, and/or leakage of the capsule by the aqueous phase, thereby promoting stability of the capsule.

In various embodiments, a capsule with the present inner layer shows significantly reduced or ablated dissolution, degradation, and/or leakage of the capsule relative to a capsule without the present inner layer.

In some embodiments, the inner layer protects the capsule from the aqueous phase, as described above, for greater than or equal to 1 day, greater than or equal to 2 days, greater than or equal to 3 days, greater than or equal to 7 days, greater than or equal to 14 days, greater than or equal to 30 days, greater than or equal to 90 days, or greater than or equal to 180 days at room temperature under ambient conditions. In certain embodiments, the inner layer protects the capsule from the aqueous phase for less than or equal to 365 days, less than or equal to 180 days, less than or equal to 90 days, less than or equal to 30 days, less than or equal to 14 days, less than or equal to 7 days, less than or equal to 3 days, or less than or equal to 2 days at room temperature under ambient conditions. Combinations of the above-referenced ranges are possible (e.g., greater than or equal to 1 day and less than or equal to 365 days). Other ranges are also possible. As such, in some embodiments, the capsule is stable at room temperature under ambient conditions for the times listed above (e.g., greater than or equal to 1 day). The phrase "ambient conditions" as used herein refers to a relative humidity of about 50%, a pressure of about 1 atm, and at room temperature, which is about 25 °C, unless specified otherwise.

In certain embodiments, the inner layer protects the capsule from the aqueous phase (e.g., the inner layer prevents the aqueous phase from contacting the capsule, prevents the aqueous phase from degrading the capsule, and/or prevents the aqueous phase from dissolving the capsule) for greater than or equal to 1 hour, greater than or equal to 2 hours, greater than or equal to 3 hours, greater than or equal to 6 hours, greater than or equal to 12 hours, greater than or equal to 18 hours, greater than or equal to 24 hours, greater than or equal to 48 hours, or greater than or equal to 96 hours at 37 °C. In certain embodiments, the inner layer protects the capsule from the aqueous phase for less than or equal to 168 hours, less than or equal to 96 hours, less than or equal to 48 hours, less than or equal to 24 hours, less than or equal to 18 hours, less than or equal to 12 hours, less than or equal to 6 hours, less than or equal to 3 hours, or less than or equal to 2 hours at 37 °C under ambient conditions. Combinations of the above-referenced ranges are possible (e.g., greater than or equal to 1 hour and less than or equal to 168 hours). As such, in certain embodiments, the capsule is stable at 37 °C under ambient conditions for the times listed above (e.g., greater than or equal to 1 hour).

Protection of the capsule from the aqueous phase may be determined by filling a capsule having an inner layer as described herein with an aqueous phase and measuring the length of time at a given temperature (e.g., room temperature and 37°C) and conditions (e.g., ambient conditions) until the aqueous phase dissolves and/or degrades at least a portion of the capsule (e.g., such that the aqueous phase leaks from the capsule). Other methods for determining whether a capsule is protected from the aqueous phase by the inner layer is described below in the section entitled "Drug Product Dissolution Assay".

In some embodiments, the inner layer comprises a material which prevents or retards the contact of the aqueous phase with the capsule (or capsule material). In certain embodiments, the inner layer comprises a hydrophobic material, i.e., which is applied as a hydrophobic coating. In some cases, the inner layer has a hydrophobic surface in contact with the aqueous phase. As used herein, the term "hydrophobic" refers to material that has a water contact angle of greater than or equal to 90 degrees. Accordingly, a "hydrophobic surface" or "hydrophobic coating" refers to a surface that has a water contact angle of greater than or equal to 90 degrees (e.g., greater than or equal to 120 degrees, greater than or equal to 150 degrees).

In embodiments, a capsule comprises an inner coating which has hydrophobic properties, which prevents or retards contact of an aqueous phase (e.g., a drug substance of the present disclosure) with the capsule (or capsule material). In embodiments, the inner coating comprises a hydrophobic coating. The hydrophobic coating may comprise a material selected from the group consisting of zein, polysaccharides, silk, polycaprolactone, oil, pectin, wax, polymers, shellac and derivatives thereof, and combinations thereof. In some embodiments, the hydrophobic coating is shellac. Non-limiting examples of suitable polysaccharides include alginate, hyaluronic acid, and chitosan. Non- limiting examples of suitable oils include avocado oil, vegetable oil, castor oil, olive oil, jojoba oil, cocoa butter, coconut oil. Non-limiting examples of suitable waxes include beeswax, carnauba wax, and paraffin wax.

A capsule of the present invention which comprises, at least, an inner layer which prevents or inhibits immediate dissolution of a capsule when filled with an aqueous phase and/or an inner layer which promotes a capsule's stability in ambient conditions is referred to herein as a "delayed-release capsule."

In some embodiments, the inner layer comprises a polymeric material. Non-limiting examples of suitable polymeric materials include polymethylmethacrylate, poly(N,N-dimethylacrylamide), polyoxamer, polyethylene glycol, polypropylene glycol, polysaccharides (e.g., sucrose, trehalose, glucose, starches such as tapioca and arrowroot, chitosan, alginate, guar gum), polyacrylate, polymethacrylate, polyvinyl alcohol, polyalkylene glycols, polyacrylamide, polyvinylpyrrolidone, polyurethane, polylactide, lactide/glycolide copolymer, polycaprolactone, polydioxanones, polyanhydride, polyhydroxybutyrate, polysiloxane, polytrimethylene carbonate, polyalkylene glycol, and combinations and/or copolymers thereof.

In certain embodiments, the inner layer comprises a polymer such as an enteric polymer. The term enteric is generally used to describe materials that are stable at relatively highly acidic pH conditions (e.g., pH of less than about 5.5) and susceptible to dissolution at relatively alkaline pH conditions (e.g., pH of between about 6 and about 9). In some embodiments, the enteric polymer is selected such that the enteric polymer is stable in an acidic gastric environment [i.e., having a pH1 to pH4) but dissolves in a more alkaline region of the gastrointestinal tract [i.e., having a pH greater than 5.5).

For example, in certain embodiments, the enteric polymer does not substantially degrade at a pH ranging between about 1 and about 5. In some embodiments, the enteric polymer does not substantially degrade at a pH of at least about 1 , at least about 2, at least about 3, at least about 4, or at least about 4.5. In certain embodiments, the enteric polymer does not substantially degrade at a pH of less than or equal to about 5, less than or equal to about 4.5, less than or equal to about 4, less than or equal to about 3, or less than or equal to about 2. Combinations of the above-referenced ranges are possible (e.g., between about 1 and about 4.5, between about 1 and about 5, between about 1 and 4). Other ranges are also possible.

In certain embodiments, the enteric polymer degrades substantially at a pH ranging between about 4 and about 8. In some embodiments, the enteric polymer degrades substantially at a pH of at least about 4, at least about 5, at least about 6, at least about 6.5, at least about 7, or at least about 7.5. In certain embodiments, the enteric polymer degrades substantially at a pH of less than or equal to about 8, less than or equal to about 7.5, less than or equal to about 7, less than or equal to about 6.5, less than or equal to about 6, or less than or equal to about 5. Combinations of the above-referenced ranges are possible (e.g., between about 4 and about 8, between about 5 and about 8, between about 6.5 and about 7.5). Other ranges are also possible.

Those skilled in the art would be capable of selecting suitable methods for determining degradation of the enteric polymers based upon the teachings of the specification including, determining the solubility of the enteric polymer in an aqueous phase having a pH of less than about 3 and/or dissolving the enteric polymer in aqueous phase having a pH of greater than or equal to about 6, measured at body temperature (e.g., at 37 °C) over a time period of between about 2 and about 40 days. In some embodiments, the enteric polymer that does not substantially degrade behaves such that less than about 10%, less than about 5%, or less than about 2% of the enteric polymer dissociates from the rest of enteric polymer. In certain embodiments, the enteric polymer that substantially degrades behaves such that at least about 1 %, at least about 2%, or at least about 5% of the enteric polymer dissociates from the remainder of the polymeric composite.

In certain embodiments, the inner layer is designed such that it releases the aqueous phase

(e.g., the inner layer dissolves and/or degrades such that the aqueous phase contacts the capsule and/or is released from the capsule) at a location within a subject, e.g., within a specific region of the digestive system of the subject.

For example, in some embodiments, the inner layer comprises a material that dissolves and/or degrades at a particular pH after a given amount of time (e.g., after at least an hour of exposure to the particular pH). In some embodiments, the inner layer is designed to release the aqueous phase (e.g., the aqueous phase comprising the microbial composition) in the colon of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 7.0 (e.g., EUDRAGIT® S 100, EUDRAGIT® S 12, 5, EUDRAGIT® FS 30D, Phloral®); see, e.g., US 9,023,368, US 20150150837, and US 20150202162. In certain embodiments, the inner layer is designed to release the aqueous phase in the duodenum of the subject and comprises an enteric polymer that at least partially dissolves and/or degrades at a pH of greater than or equal to 5.5 (e.g., EUDRAGIT® L 30D-55, EUDRAGIT® L 100-55). In some embodiments, the inner layer is designed to release the aqueous phase in the jejunum of the subject and comprises an enteric polymer that at least partially dissolves and/or degrades at a pH of greater than or equal to 6.0 and less than or equal to 7.0 (e.g., EUDRAGIT® L 100, EUDRAGIT® L 12, 5). In certain embodiments, the inner layer is designed to release the aqueous phase in the stomach of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 1 .0 and less than or equal to 5.0 (e.g., EUDRAGIT® E 100, EUDRAGIT® E 12, 5, EUDRAGIT® E PO).

In some embodiments, the enteric polymer is selected such that it exhibits time controlled degradation and/or dissolution, independent of pH . Non-limiting examples of such enteric polymers include EUDRAGIT® RL 30D, EUDRAGIT® RL PO, EUDRAGIT® RL 100, EUDRAGIT® RL 12, 5, EUDRAGIT® RS 30D, EUDRAGIT® RS PO, EUDRAGIT® RS 100, EUDRAGIT® RS 12, 5, EUDRAGIT® NE 30D, EUDRAGIT® NE 40D, and EUDRAGIT® NM 30D.

In certain embodiments, the polymer is a copolymer of two or more enteric polymers described herein.

In some embodiments, the enteric polymer includes, but is not limited to, cellulose acetate phthalate (CAP), hypromellose (INN) hydroxypropyl methylcellulose (HPMC), poly(methacrylic acid-co- ethyl acrylate) (e.g., EUDRAGIT®, available from Evonik Industries AG (Essen, Germany)), derivatives thereof, and copolymers thereof.

In some embodiments, the inner layer comprises an enteric elastomer. In some embodiments, the enteric elastomer comprises a mixture of two or more polymers with carboxyl functionality such that the two or more polymers form hydrogen bonds with one another and has both enteric and elastic properties. In certain embodiments, the enteric elastomer comprises a first polymer comprising a structure as in Formula (I):

In some embodiments, the inner layer comprises a polymer formed by the reaction of one or more monomers in the presence of a food grade catalyst (e.g., caffeine). Suitable polymers formed in the presence of food grade catalysts are described in more detail in International Patent Publication No. WO 2015/168297, entitled "Polymeric Materials for Bio-Applications," published on November 5, 2015, and filed on April 29, 2015 as International Patent Application Serial No. PCT/US 15/28311 , which is incorporated herein by reference in its entirety for all purposes. Outer Layer

In some embodiments, the article comprises an outer layer. In certain embodiments, the outer layer separates the external environment from the capsule, thereby inhibiting dissolution of a capsule and/or promoting stability of the capsule. In embodiments, the article comprises two or more outer layers which are sequentially applied, e.g., after a first outer layer has been applied and air dried, a second outer layer is applied. Unless the context clearly dictates otherwise, as used herein, the term "outer layer" includes at least one outer layer, e.g., one, two, three, four, or more outer layers. Moreover, a first outer layer may comprise identical components as an at least second outer layer or a first outer layer may comprise different components from an at least second outer layer.

Illustrated in FIG. 2, article 100 comprises an outer layer 140 adjacent capsule 130. In certain embodiments, outer layer 140 is directly adjacent capsule 130. Article 100 further comprises an inner layer 120 which separates the aqueous phase 1 10 from the capsule 130.

In some embodiments, the outer layer comprises a delayed release material such that the microbial composition released from the capsule at a location internally to a subject. Those skilled in the art would understand based upon the teachings of this specification that an article (e.g., comprising a capsule, an inner layer, and/or an outer layer) which is designed to release a microbial composition from the article at a location internally to a subject may also be designed to not release the microbial composition prior the article reaching the location internally to the subject.

A capsule of the present invention which comprises, at least, an outer layer which prevents or inhibits immediate dissolution of a capsule in an aqueous medium and/or an outer layer which promotes a capsule's stability in ambient conditions is referred to herein as a "delayed-release capsule."

In certain embodiments, the outer layer may comprise a bioadherent polymer such as mucin. In some embodiments, the outer layer comprises a polymeric material. Non-limiting examples of suitable polymeric materials include gelatin, polymethylmethacrylate, poly(N,N-dimethylacrylamide), polyoxamer, polyethylene glycol, polypropylene glycol, polysaccharides (e.g., sucrose, trehalose, glucose, starches such as tapioca and arrowroot, chitosan, alginate, guar gum), polyacrylate, polymethacrylate, polyvinyl alcohol, polyalkylene glycols, polyacrylamide, polyvinylpyrrolidone, polyurethane, polylactide, lactide/glycolide copolymer, polycaprolactone, polydioxanones, polyanhydride, polyhydroxybutyrate, polysiloxane, polytrimethylene carbonate, polyalkylene glycol, and combinations and/or copolymers thereof.

In certain embodiments, the outer layer comprises a polymer such as an enteric polymer. In some embodiments, the enteric polymer includes, but is not limited to, cellulose acetate phthalate (CAP), hypromellose (INN) hydroxypropyl methylcellulose (HPMC), poly(methacrylic acid-co-ethyl acrylate) (e.g., EUDRAGIT®, available from Evonik Industries AG (Essen, Germany)), derivatives thereof, and copolymers thereof.

In some embodiments, the outer layer comprises a material that dissolves and/or degrades at a particular pH after a given amount of time (e.g., after at least an hour of exposure to the particular pH). In some embodiments, the outer layer is designed to at least partially degrade and/or at least partially dissolve in the colon of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 7.0 (e.g., EUDRAGIT® S 100, EUDRAGIT® S 12, 5, EUDRAGIT® FS 30D, Phloral®); see, e.g., US 9,023,368, US 20150150837, and US 20150202162.

In certain embodiments, the outer layer is designed to at least partially dissolve and/or at least partially degrade in the duodenum of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 5.5 (e.g., EUDRAGIT® L 30D-55, EUDRAGIT® L 100-55). In some embodiments, the outer layer is designed to at least partially dissolve and/or at least partially degrade in the jejunum of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 6.0 and less than or equal to 7.0 (e.g., EUDRAGIT® L 100, EUDRAGIT® L 12, 5). In certain embodiments, the outer layer is designed to at least partially dissolve and/or at least partially degrade in the stomach of the subject and comprises an enteric polymer that at least partially dissolves and/or at least partially degrades at a pH of greater than or equal to 1 .0 and less than or equal to 5.0 (e.g., EUDRAGIT® E 100, EUDRAGIT® E 12, 5, EUDRAGIT® E PO).

In some embodiments, the enteric polymer is selected such that it exhibits time controlled degradation and/or dissolution, independent of pH. Non-limiting examples of such enteric polymers include EUDRAGIT® RL 30D, EUDRAGIT® RL PO, EUDRAGIT® RL 100, EUDRAGIT® RL 12, 5, EUDRAGIT® RS 30D, EUDRAGIT® RS PO, EUDRAGIT® RS 100, EUDRAGIT® RS 12, 5, EUDRAGIT® NE 30D, EUDRAGIT® NE 40D, and EUDRAGIT® NM 30D.

In certain embodiments, the outer layer comprises a copolymer of two or more enteric polymers described herein.

In some embodiments, the outer layer comprises an enteric elastomer. In some embodiments, the enteric elastomer comprises a mixture of two or more polymers with carboxyl functionality such that the two or more polymers form hydrogen bonds with one another and has both enteric and elastic properties. In certain embodiments, the enteric elastomer comprises a first polymer comprising a structure as in Formula (I):

In some embodiments, the outer layer comprises a polymer formed by the reaction of one or more monomers in the presence of a food grade catalyst (e.g., caffeine). Suitable polymers formed in the presence of food grade catalysts are described in more detail in International Patent Publication No. WO 2015/168297, entitled "Polymeric Materials for Bio-Applications," published on November 5, 2015, and filed on April 29, 2015 as International Patent Application Serial No. PCT/US 15/28311 , which is incorporated herein by reference in its entirety for all purposes.

Using Phloral® as an exemplary outer coating, a solution of EUDRAGIT® S100

(poly(methacrylic acid, methylmethacrylate)), Starch, Triethyl Citrate, and PlasACRYL T20 dissolved in a solution of water, ethanol, and n-butanol are mixed and then charged to a Fluid Bed Coater. The solution is then spray coated on the outer surface of the capsule bodies and capsule caps to a target weight gain. The capsule bodies and capsule caps are allowed to dry for about 8 hours to about 24 hours, or longer, e.g., for a week, a month, or more, before further procession, e.g., adding an inner coat and filling with drug product. Other outer coating compositions, as described herein and as known in the art, may be used to apply an outer coating.

Manufacturing Process of a Capsule

Generally, manufacturing a capsule of the present invention (e.g., a delayed-release capsule) may comprise steps of: (1) coating the exterior of a dissociated capsule {i.e., comprising separate capsule body and capsule cap, i.e., the capsule precursors) with the outer layer, (2) coating the interior of the dissociated capsule [i.e., the capsule body and the capsule cap) with the inner layer, (3) providing the capsule body with a drug product, and (4) closing the capsule cap over the capsule body, thereby encapsulation of the drug product in a dual-coated capsule. In embodiments of such dual- coated capsules, the inner layer may be applied before the outer layer is applied. Manufacturing a capsule of the present invention may alternately comprise steps of: (1) coating the interior of dissociated capsule [i.e., comprising a separate capsule body and capsule cap, also referred herein as a "capsule precursor") with the inner layer, (2) providing the capsule body with a drug product, and (3) closing the capsule cap over the capsule body, thereby encapsulation the drug product in a capsule; this embodiment may be used when only a single (inner) layer is needed to achieve desired release profiles and/or when a capsule comprises enteric/delayed-release components.

In embodiments, it may be desirable to provide an amount of drug product to a capsule's cap, in addition to providing the drug product to the capsule's body. In this embodiment, more drug product will be included in a capsule and/or less air will be contained in a closed capsule.

The articles described herein may be manufactured using any suitable method. For example, in some embodiments, a capsule (e.g., a capsule comprising an inner layer) is filled with an aqueous phase comprising a microbial composition and then closed/sealed such that the aqueous phase is encapsulated by the capsule. In certain embodiments, the capsule is coated externally with an outer layer (e.g., comprising an enteric polymer) after filling the capsule with the aqueous phase. However, in some cases, coating the capsule after filling may not be desirable as the coating process may, for example reduce viability of the microbial composition (e.g., as a result of the use of solvents and/or relatively high temperatures during the coating process) as compared to the viability of the microbial composition prior to coating .

As such, in some embodiments, the capsule if formed from a capsule precursor which has been coated externally with an outer layer. Advantageously, the coating of a capsule precursor prior to filling the capsule (or capsule precursor) with a microbial composition may substantially retain the viability of the microbial composition. In some cases, other compositions for filling the capsule may be possible. For example, compositions in which the coating process affects viability, efficacy, concentration, and/or toxicity of the composition may be incorporated into a capsule using a precursor capsule as described herein.

Additionally, coated capsule precursors are stable, e.g., over a year to indefinitely, in ambient conditions and/or in a sealed storage container. Thus, coated capsule precursors can be produced in advance of the time of filling capsules.

In certain embodiments, a capsule may be formed from one or more capsule precursors. As illustrated in FIG. 3A, in some embodiments, a capsule precursor 300 comprises a capsule portion 330 having an external surface 332 and an internal surface 334. Those skilled in the art would understand, based upon the teachings of this specification, that the capsule precursor is not intended to refer to a capsule which has been closed, coated with an outer layer, and then reopened (e.g., such that the capsule may be filled). By contrast, the one or more capsule precursors are coated prior to any closing of the capsule (and/or filling of the capsule with an aqueous phase).

As illustrated in FIG. 3B, in certain embodiments, capsule precursor 300 may be coated externally with an outer layer 340. For example, in some embodiments, outer layer 340 is coated on at least a portion of external surface 332 of capsule portion 330. In some embodiments, substantially all of external surface 332 is coated with outer layer 340. In certain embodiments, at least a portion of internal surface 334 may also be coated with the outer layer. However, in some embodiments, substantially none of the outer layer material is present on internal surface 334.

In a preferred embodiment, the bodies and caps of gelatin capsules (e.g., size #00) are separate. An outer enteric coating suspension is prepared by dispersing one or more enteric coating polymers in a solution. The outer enteric coating suspension is applied to the exterior of the separate capsule bodies and caps in two-process runs using a Fluid Bed Coater (or an equivalent). The capsule bodies and caps are sprayed with the enteric coating suspension, e.g., the exemplary Phloral® outer coating described above or another outer coating compositions, as described herein or as known in the art, until an outer coating of a target weight, e.g., between about 2 mg/cm² and 6 mg/cm² in certain embodiments, is achieved. The capsules are then set out until dry, e.g., between about 8 hours and 24 hours. After drying, exemplary capsules may be weighed to calculate weight gain from the outer enteric coating. Capsules may be inspected for irregularities.

Capsule portion 330, at internal surface 334, may be configured to receive and/or retain a fluid such as an aqueous phase. In some embodiments, the fluid (e.g., the aqueous phase) comprises a microbial composition. In certain embodiments, the fluid comprises a therapeutic agent. In some cases, capsule precursor 300 may be closed/sealed, forming a capsule, and such that a fluid (e.g., comprising a therapeutic agent and/or a microbial composition) is encapsulated by the formed capsule. In some embodiments, two capsule portions may be joined together forming the capsule.

The capsule portion may comprise any material suitable for a capsule (e.g., a degradable material) as described herein. The outer layer may comprise any suitable material for coating a capsule (e.g., an enteric polymer) as described herein. For example, the outer layer may comprise a pH- sensitive material such that the capsule may be administered to a subject and the encapsulated fluid in the capsule released at a location internally to the subject, as described herein.

Capsule portion 330, at internal surface 334, may be configured to receive and/or retain a fluid such as an aqueous phase. In some embodiments, the fluid (e.g., the aqueous phase) comprises a microbial composition. In certain embodiments, the fluid comprises a therapeutic agent. In some cases, capsule portion 330 may be closed/sealed, forming a capsule, and such that a fluid (e.g., comprising a therapeutic agent and/or a microbial composition) is encapsulated by the formed capsule. In some embodiments, two capsule portions may be joined together forming the capsule.

In an exemplary embodiment, a capsule may be formed by coating at least an external surface of a capsule portion with an outer layer, prior to filling the capsule with a fluid (e.g., an aqueous phase). In certain embodiments, after coating at least an external surface of the capsule portion with the outer layer, the capsule portion may be filled with the fluid and closed/sealed such that the fluid is encapsulated, and thus forming a capsule.

In some embodiments, the drug product (and/or an additional therapeutic agent) may be reduced in efficacy, concentration, uptake, and/or viability if a capsule were coated with an outer layer after the capsule is filled with the drug product (and/or an additional therapeutic agent).

In certain embodiments, the capsule precursor comprises an inner layer. For example, as illustrated in FIG. 3C, internal surface 334 of capsule portion 330 may be coated with an inner layer 320 (e.g., an inner layer selected and designed such that it protects the capsule (or capsule portion) from the aqueous phase) prior to filling and/or closing/sealing the capsule.

In some embodiments outer layer 340 is adjacent capsule portion 330. In certain embodiments, outer layer 340 is directly adjacent capsule portion 330. In some cases, inner layer 320 may be adjacent (e.g., directly adjacent) capsule portion 330.

In embodiments, capsule bodies and capsule caps comprising an outer layer coating are then provided an inner surface coating. Alternately, capsule bodies and capsule caps lacking an outer layer, i.e., uncoated capsule precursors, may be provided an inner coating.

The interior surfaces of uncoated capsules precursors or outer-coated capsule precursors are then provided an internal coating. As an example, a hydrophobic coating (e.g., shellac) is prepared in a sanitized biosafety cabinet (BSC). In embodiments, the hydrophobic coating is provided as a solution comprising a liquid, e.g., an alcohol, and the hydrophobic coating, e.g., shellac. The ratio of shellac to alcohol, e.g., ethanol, can vary from 0.1 g shellac per ml ethanol to 1 .3 g shellac per ml of ethanol, e.g., 0.7, 0.8, 0.9, and 1.0 g shellac per ml ethanol. At the higher end of the range, the solution may be too viscous for efficient use and/or the shellac may be too abundant to get into solution. In some embodiments, the internal coating is a 1 :1 solution of shellac and ethanol [i.e., 1 g shellac per 1 ml ethanol).

The hydrophobic coating solution is dispensed into the capsule bodies and the capsule caps to create a film of the hydrophobic coating on the inner surface of the capsule precursor. In embodiments using a size 00 capsule, between about 700 μΙ to about 1000 μΙ of the hydrophobic coating solution, e.g., about 700 μΙ, about 750 μΙ, about 800 μΙ, about 850 μΙ, about 900 μΙ, about 950 μΙ, about 1000 μΙ, and up to the capsule body's full capacity, is dispensed per capsule body. In embodiments using a size 00 capsule, about 50 μΙ to about 400 μΙ of the hydrophobic coating solution, e.g., about 50 μΙ, about 75 μΙ, about 100 μΙ, about 125 μΙ, about 150 μΙ, about 175 μΙ, about 200 μΙ, about 225 μΙ, about 250 μΙ, about 275 μΙ, about 300 μΙ, about 325 μΙ, about 350 μΙ, about 375 μΙ, and about 400 μΙ, is dispensed per capsule cap. The volume of dispensed solution depends on the capsule's size. The capsule bodies may be filled entirely. However, in some embodiments, the capsule cap is not entirety filled; thus, there will be a ring around the internal surface of the open end of the capsule cap which does not comprise an inner layer. This ring will allow a gentle seal to form between capsule cap and the capsule body. Absent the ring, the excess material on the open end of the capsule may prevent sealing of the capsule cap onto the capsule body or may require additional force to enact a seal. It is within the ability of a skilled artisan to determine the volume of solution to be dispensed into a capsule cap such that an above-described ring is sufficiently formed.

After applying an inner layer solution, the bodies and caps are typically allowed to dry, e.g., air dry, for up to about three days, e.g., twenty-four hours, two days, or longer. In embodiments, bodies and caps are allowed to dry for one week, for one month, or for longer. After drying, exemplary capsules are weighed to calculate weight gain from the hydrophobic coating. Capsules may be inspected for irregularities.

In embodiments, capsule caps and capsule bodies are each provided an inner coating of a 1 :1 solution of shellac and ethanol.

The inner surface-alone coated capsule bodies or dual-coated capsule bodies are then filled with a drug product (as described herein). Here, inner-coated-alone capsule bodies or dual-coated capsule bodies are arranged in capsule capping racks and are filled with about 750 μΙ_ of drug product for a #00 capsule (e.g., about 500 μΙ, about 550 μΙ, about 600 μΙ, about 650 μΙ, about 700 μΙ, about 750 μΙ, about 800 μΙ, about 850 μΙ, about 900 μΙ, about 950 μΙ, about 1000 μΙ, and up to the capsule body's full capacity), using a multichannel pipetting system and sterile filter tips. Filled capsule bodies are capped with outer-coated capsule caps, thereby producing a (filled) delayed-release capsule of the present invention.

From each lot of filled delayed-release capsules, the first, last, and every 100th capsule may be weighed. For example, a dual-coated #00 capsules filed with about 750 μΙ_ of drug substance should weigh between 0.88 and 1.16 grams. Should the three capsules have weights outside this range, then each capsule in the lot is weighed. Capsules may be visually sorted for integrity and for visible surface irregularities or discolorations; capsules failing this visual inspection may be discarded.

In various embodiments, the capsule comprises at least one (e.g., one, two, three, or four) outer enteric coatings and/or at least one (e.g., one, two, three, or four) inner coatings. In various embodiments, the inner coating is a hydrophobic coating. In various embodiments, the at least one outer enteric coating is provided before the at least one inner coating is provided or the at least one outer enteric coating is provided after the at least one inner coating is provided. Alternately, inner surface-alone coated capsules only comprise at least one inner coating and lack an outer coating.

In various embodiments, the coating is between about 2 mg/cm² and 6 mg/cm², e.g., about 3 mg/cm².

A capsule of the present invention and comprising an inner layer and/or comprising an outer layer may be referred to herein as a delayed-release capsule.

Any of the above-described inner layers, capsules, and outer layers may be combined into a delayed-release capsule of the present invention. A skilled artisan would know how to select an inner layer, a capsule, and an outer layer according to his/her present need, which would be based on the specific drug product and/or the location in a subject (e.g., in the gastrointestinal system) where the drug product should be released.

In some embodiments, one or more of the inner layer, the capsule, and/or the outer layer comprise only non-toxic materials. For example, in certain embodiments, one or more of the inner layer, the capsule, and/or the outer layer include substantially no materials other than those included on the FDA's "Generally Recognized as Safe" Substances database and/or listed in 21 C.F.R. § 182.

The term "toxic" refers to a substance showing detrimental, deleterious, harmful, or otherwise negative effects on a subject, tissue, or cell when or after administering the substance to the subject or contacting the tissue or cell with the substance, compared to the subject, tissue, or cell prior to administering the substance to the subject or contacting the tissue or cell with the substance. In certain embodiments, the effect is death or destruction of the subject, tissue, or cell. In certain embodiments, the effect is a detrimental effect on the metabolism of the subject, tissue, or cell. In certain embodiments, a toxic substance is a substance that has a median lethal dose (LD50) of not more than 500 milligrams per kilogram of body weight when administered orally to an albino rat weighing between 200 and 300 grams, inclusive. In certain embodiments, a toxic substance is a substance that has an LD50 of not more than 1 ,000 milligrams per kilogram of body weight when administered by continuous contact for 24 hours (or less if death occurs within 24 hours) with the bare skin of an albino rabbit weighing between two and three kilograms, inclusive. In certain embodiments, a toxic substance is a substance that has an LC50 in air of not more than 2,000 parts per million by volume of gas or vapor, or not more than 20 milligrams per liter of mist, fume, or dust, when administered by continuous inhalation for one hour (or less if death occurs within one hour) to an albino rat weighing between 200 and 300 grams, inclusive.

The term "non-toxic" refers to a substance that is not toxic. Toxic compounds include, e.g., oxidative stressors, nitrosative stressors, proteasome inhibitors, inhibitors of mitochondrial function, ionophores, inhibitors of vacuolar ATPases, inducers of endoplasmic reticulum (ER) stress, and inhibitors of endoplasmic reticulum associated degradation (ERAD). In some embodiments a toxic agent selectively causes damage to nervous system tissue. Toxic compounds include compounds that are directly toxic and agents that are metabolized to or give rise to substances that are directly toxic. It will be understood that the term "toxic compounds" typically refers to compounds that are not ordinarily present in a cell's normal environment at sufficient levels to exert detectable damaging effects. However, in some cases, the toxic compounds may be present in a cell's normal environment but at concentrations significantly less than present in the auxiliary materials described herein. Typically toxic compounds exert damaging effects when present at a relatively low concentration, e.g., at or below 1 mM, e.g., at or below 500 μΜ, e.g., at or below 100 μΜ . It will be understood that a toxic compound typically has a threshold concentration below which it does not exert detectable damaging effects. The particular threshold concentration will vary depending on the agent and, potentially, other factors such as cell type, other agents present in the environment.

In some embodiments, one or more components of the article (e.g., the capsule, the inner layer, the outer layer, the aqueous phase) includes one or more additives. In some embodiments, the additive is selected from the group consisting of starches, genipin, aloe, whey protein, guar gum, xantham gum, polyethylene glycol, and carrageenan. Non-limiting examples of suitable starches include amylose, tapioca, and arrowroot. One or more additives may be added to the article (or a component of the article) to, for example, increase stability under a particular pH, temperature, and/or humidity. In some cases, the one or more additives may increase the mechanical properties of the article (or a component of the article).

In some embodiments, the additive comprises a preservative. For example, in some embodiments, the aqueous phase comprises a microbial composition and a preservative such as glycerin. Other preservatives are also possible. Delayed-Release Capsules

In some embodiments, the articles described herein are administered to a subject. In certain embodiments, the articles are designed such that a microbial composition contained therein is not released from the article until reaching a location internally of a subject, where upon the microbial composition is released from the article. In some embodiments, the microbial composition is released from the article via degradation and/or dissolution (e.g., by local pH and solutions present at the location internally of the subject) of at least a portion of the capsule, the inner layer, and/or the outer layer. In some embodiments, the article is administered to a subject orally. In certain embodiments, the article may be administered orally, rectally, vaginally, nasally, or uretherally.

In some embodiments, the delayed-release capsules described herein are designed for targeted release of a drug product (e.g., a microbial composition) to a selected location internally of a subject. In some embodiments, the location internally of the subject is the colon, the duodenum, the ileum, the colon, the duodenum, the jejunum, the stomach, or the esophagus.

In an illustrative embodiment, an article is designed such that the microbial composition encapsulated therein is released in the colon of a subject (e.g., a location internally of the subject). The article comprises an inner layer adjacent a capsule that protects the capsule from an encapsulated aqueous phase (comprising the microbial composition) and an outer layer. The outer layer comprises a material (e.g., an enteric polymer) that selectively degrades and/or dissolves at a pH of greater than 7.0 such that the microbial composition is released from the article in the colon of the subject. In some embodiments, the inner layer also comprises a material that selectively degrades and/or dissolves at a pH of greater than 7.0. The article, prior to administration, is stable at room temperature for at least 1 day at room temperature such that the microbial composition is not released from the article for at least 1 day. In some embodiments, the article is administered orally (e.g., the subject swallows the article) and the article transits the gastrointestinal tract without releasing the microbial composition until reaching the selected location internally of the subject such as the colon.

The term "subject," as used herein, refers to an individual organism such as a human or an animal. In some embodiments, the subject is a mammal (e.g., a human, a non-human primate, or a non- human mammal), a vertebrate, a laboratory animal, a domesticated animal, an agricultural animal, or a companion animal. In some embodiments, the subject is a human (e.g., a human patient). In some embodiments, the subject is a rodent, a mouse, a rat, a hamster, a rabbit, a dog, a cat, a cow, a goat, a sheep, or a pig.

In various embodiments, a capsule may be manufactured to produce a delayed-release, i.e., slow release of drug product, e.g., a microbial composition or FMT composition, in the body (e.g., Gl tract) over an extended period of time. In various embodiments, the capsule does not immediately release the drug product upon ingestion; rather, postponement of the release of the drug substance until the capsule is lower in the Gl tract; for example, for release in the small intestine (e.g., one or more of duodenum, jejunum, ileum) or the large intestine (e.g., one or more of cecum, ascending, transverse, descending or sigmoid portions of the colon, and rectum). For example, a capsule can be coated, as described herein, to delay release of the drug product until it reaches the small intestine and/or the large intestine. In various embodiments, a capsule may utilize one or more delayed-release coatings such as delayed-release coatings to provide for effective, delayed yet substantial delivery of the drug product to the Gl tract and, optionally, with additional therapeutic agents.

Various methods may be used to deliver the drug product, described herein, to a location of interest. For example, the capsule may be formulated for delivery of the drug product (with or without additional therapeutic agents) to the Gl tract. The Gl tract includes organs of the digestive system such as mouth, esophagus, stomach, duodenum, small intestine, large intestine (also referred here to as the "colon") and rectum and includes all subsections thereof (e.g., the small intestine may include the duodenum, jejunum and ileum; the large intestine may include the colon transversum, colon descendens, colon ascendens, colon sigmoidenum and cecum). For example, the capsule herein may be formulated for delivery of a drug product (with our without additional therapeutic agents) to one or more of the stomach, small intestine, large intestine and rectum and includes all subsections thereof (e.g., duodenum, jejunum and ileum, colon transversum, colon descendens, colon ascendens, colon sigmoidenum and cecum). In some embodiments, the capsule described herein may be formulated to deliver to the upper or lower Gl tract.

For example, in various embodiments, the present invention provides delayed-release capsules which release a substantial amount of the drug substance (with or without additional therapeutic agents) into one or more regions of the Gl tract. For example, at least about 60% of the drug product may be released after the stomach and into one or more regions of the Gl tract.

In various embodiments, the delayed-release capsule of the present invention release at least

60% of the drug product (and/or additional therapeutic agents) after the stomach into one or more regions of the intestine. For example, delayed-release capsule of the present invention release at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the drug product (and/or additional therapeutic agents) in the intestines.

In various embodiments, delayed-release capsule of the present invention release at least 60% of the drug product (and/or additional therapeutic agents) in the small intestine. For example, the delayed-release capsule releases at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the drug product (and/or additional therapeutic agents) in the small intestine (e.g., one or more of duodenum, jejunum, ileum, and ileocecal junction).

In various embodiments, the delayed-release capsule of the present invention releases at least 60% of the drug product (and/or additional therapeutic agents) in the large intestine. For example, the delayed-release capsule releases at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the drug product (and/or additional therapeutic agents) in the large intestine (e.g., one or more of cecum, ascending, transverse, descending or sigmoid portions of the colon, and rectum).

In various embodiments, the delayed-release capsule provides substantially complete delivery of the drug product (and/or additional therapeutic agents) prior to the rectum.

In some embodiments, the delayed-release capsule releases the drug product (and/or additional therapeutic agents) in the stomach. In other embodiments, the delayed-release capsule is formulated so as to not substantially release the drug product (and/or additional therapeutic agents) in the stomach.

In certain embodiments, the delayed-release capsule releases the drug substance (and/or additional therapeutic agents) at a specific pH. For example, in some embodiments, the delayed-release capsule is substantially stable in an acidic environment and substantially unstable (e.g., dissolves rapidly or is physically unstable) in a near neutral to alkaline environment. In some embodiments, stability is indicative of not substantially releasing while instability is indicative of substantially releasing. For example, in some embodiments, the delayed-release capsule is substantially stable at a pH of about 7.0 or less, or about 6.5 or less, or about 6.0 or less, or about 5.5 or less, or about 5.0 or less, or about 4.5 or less, or about 4.0 or less, or about 3.5 or less, or about 3.0 or less, or about 2.5 or less, or about 2.0 or less, or about 1 .5 or less, or about 1 .0 or less. In some embodiments, the present formulations are stable in lower pH areas and therefore do not substantially release drug product (and/or additional therapeutic agents) in, for example, the stomach. In some embodiments, delayed-release capsule is substantially stable at a pH of about 1 to about 4 or lower and substantially unstable at pH values that are greater. In these embodiments, the delayed-release capsule does not substantially release drug product (and/or additional therapeutic agents) in the stomach. In these embodiments, the delayed- release capsule substantially releases drug product (and/or additional therapeutic agents) in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) and/or large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon). In some embodiments, delayed-release capsule is substantially stable at a pH of about 4 to about 5 or lower and consequentially is substantially unstable at pH values that are greater and therefore, the drug product (and/or additional therapeutic agents) is not substantially released in the stomach and/or small intestine (e.g., one or more of the duodenum, jejunum, and ileum). In these embodiments, the delayed-release capsule substantially releases drug product (and/or additional therapeutic agents) in the large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon). In various embodiments, the pH values recited herein may be adjusted as known in the art to account for the state of the subject, e.g., whether in a fasting or postprandial state.

In some embodiments, the delayed-release capsule is substantially stable in gastric fluid and substantially unstable in intestinal fluid and, accordingly, the drug product (and/or additional therapeutic agents) is substantially released in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) and/or large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon).

In some embodiments, the delayed-release capsule is stable in gastric fluid or stable in acidic environments. These delayed-release capsules release about 30% or less by weight of their drug products (and/or additional therapeutic agents) in gastric fluid with a pH of about 4 to about 5 or less, or simulated gastric fluid with a pH of about 4 to about 5 or less, in about 15, or about 30, or about 45, or about 60, or about 90 minutes. Delayed-release capsules of the of the invention may release from about 0% to about 30%, from about 0% to about 25%, from about 0% to about 20%, from about 0% to about 15%, from about 0% to about 10%, about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10% by weight of its drug product (and/or additional therapeutic agents) in gastric fluid with a pH of 4-5, or less or simulated gastric fluid with a pH of 4-5 or less, in about 15, or about 30, or about 45, or about 60, or about 90 minutes. Delayed-release capsule of the invention may release about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight of its drug product (and/or additional therapeutic agents) in gastric fluid with a pH of 5 or less, or simulated gastric fluid with a pH of 5 or less, in about 15, or about 30, or about 45, or about 60, or about 90 minutes.

In some embodiments, the delayed-release capsule is unstable in intestinal fluid. These delayed-release capsule release about 70% or more by weight of its drug product (and/or additional therapeutic agents) in intestinal fluid or simulated intestinal fluid in about 15, or about 30, or about 45, or about 60, or about 90 minutes. In some embodiments, the delayed-release capsule is unstable in near neutral to alkaline environments. These delayed-release capsules release about 70% or more by weight of their drug products (and/or additional therapeutic agents) in intestinal fluid with a pH of about 4-5 or greater, or simulated intestinal fluid with a pH of about 4-5 or greater, in about 15, or about 30, or about 45, or about 60, or about 90 minutes. A delayed-release capsule that is unstable in near neutral or alkaline environments may release 70% or more by weight of their drug product (and/or additional therapeutic agents) in a fluid having a pH greater than about 5 (e.g., a fluid having a pH of from about 5 to about 14, from about 6 to about 14, from about 7 to about 14, from about 8 to about 14, from about 9 to about 14, from about 10 to about 14, or from about 1 1 to about 14) in from about 5 minutes to about 90 minutes, or from about 10 minutes to about 90 minutes, or from about 15 minutes to about 90 minutes, or from about 20 minutes to about 90 minutes, or from about 25 minutes to about 90 minutes, or from about 30 minutes to about 90 minutes, or from about 5 minutes to about 60 minutes, or from about 10 minutes to about 60 minutes, or from about 15 minutes to about 60 minutes, or from about 20 minutes to about 60 minutes, or from about 25 minutes to about 90 minutes, or from about 30 minutes to about 60 minutes.

Examples of simulated gastric fluid and simulated intestinal fluid include, but are not limited to, those disclosed in the 2005 Pharmacopeia 23NF/28USP in Test Solutions at page 2858 and/or other simulated gastric fluids and simulated intestinal fluids known to those of skill in the art, for example, simulated gastric fluid and/or intestinal fluid prepared without enzymes.

In various embodiments, the delayed-release capsule of the invention is substantially stable in chyme. For example, there is, in some embodiments, a loss of less about 50% or about 40%, or about 30%, or about 20%, or about 10% of their drug product (and/or additional therapeutic agents) in about 10, or 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 hour from administration.

Drug Product Dissolution Assay

The delayed-release capsule of the present invention comprises delayed-release coatings such that a contained drug product is released under specified, desired conditions which are found in certain locations within the human gastrointestinal (Gl) tract. Below are described exemplary steps for determining whether or not a drug product will be released from a delayed-release capsule under the specified, desired conditions.

In some embodiments, delayed-release capsules are placed into dissolution baths which mimic the osmolality, pH, humidity, motion, and/or temperature of different environments in the Gl tract. Samples are taken from the baths at varying time points to determine if any delayed-release capsule have lost their mechanical integrity and are releasing the drug product. In this assay, certain steps are performed as described in United States Pharmacopeia (USP) "<711 > Dissolution" (February 1 , 2012); the contents of which are incorporated herein by reference in their entirety.

Dissolution of individual delayed-release capsule is conducted by the following steps (multiple capsules may be run through the assay in parallel). First, one delayed-release capsule is placed in simulated gastric fluid (SGF) with pepsin at pH 1 .2 according to USP and agitated vigorously for approximately three hours, thereby mimicking residence in the stomach. Then, the SGF is replaced with simulated intestinal fluid (SIF) with pancreatin at pH 6.8 according to USP; the capsule is lightly agitated for approximately two hours, thereby mimicking transit through the small intestine. Finally, the pH of the SIF is adjusted to 7.4; the capsules are lightly agitated for approximately two hours, thereby mimicking passage into the colon. Since the delayed-release capsules of the present invention are designed for enteric release, mechanical disintegration and detectable levels of drug product release should occur about two hours after the SIF was adjusted to pH 7.4, when conditions mimic passage to the upper colon. In embodiments, the, delayed-release capsules are filled with an aqueous phase having a pH, osmolality, and/or viscosity equivalent to a desired drug product (e.g., an aqueous phase comprising glycerol) and comprising a detectable substance, e.g., food coloring.

In some embodiments, delayed-release capsules are tested for their ability to contain an aqueous drug product over an extended period of time. Here, a delayed-release capsule comprising at least an inner layer coating, is filled with an aqueous phase comprising a color dye, e.g., food coloring. In embodiments, the aqueous phase comprises a pH, osmolality, and/or viscosity equivalent to a desired drug product, e.g., an aqueous phase comprising glycerol. The filled capsule is left at ambient conditions and leakage of the colored dye is visually inspected. The phrase "ambient conditions" as used herein refers to a relative humidity of about 50%, a pressure of about 1 atm, and at room temperature, which is about 25 °C, unless specified otherwise.

It is well-known in the art that such an aqueous phase will leak from an uncoated gelatin capsule within 5 to 10 minutes. On the other hand, certain capsules of the present invention had no substantial leakage after two weeks at ambient.

Additionally, as described below, uncoated gelatin capsules place in a simulated SGF (pH 1 .2) began to exhibit aqueous phase leakage after 5 minutes, and the capsules were completely depleted after three hours. On the other hand, capsules of the present invention are able to resist aqueous phase leakage and only lost 15% of their contents after three hours in SGF.

Diseases/Disorders Treated by Drug Product of the Present Invention

The drug products of the present invention are used to treat diseases/disorders associated with the presence of abnormal enteric microflora. Such diseases/disorders include but are not limited to those conditions in the following categories: gastro-intestinal disorders including irritable bowel syndrome or spastic colon, functional bowel disease (FBD), including constipation predominant FBD, pain predominant FBD, upper abdominal FBD, nonulcer dyspepsia (NUD), gastro-esophageal reflux, inflammatory bowel disease including Crohn's disease, ulcerative colitis, indeterminate colitis, collagenous colitis, microscopic colitis, chronic Clostridium difficile infection, pseudemembranous colitis, mucous colitis, antibiotic associated colitis, idiopathic or simple constipation, diverticular disease, AIDS enteropathy, small bowel bacterial overgrowth, coeliac disease, polyposis coil, colonic polyps, chronic idiopathic pseudo obstructive syndrome, and toxic megacolon; chronic gut infections with specific pathogens including bacteria, viruses, fungi, and protozoa; viral gastrointestinal disorders, including viral gastroenteritis, Norwalk viral gastroenteritis, rotavirus gastroenteritis, and AIDS related gastroenteritis; liver disorders such as primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver, and cryptogenic cirrhosis; rheumatic disorders such as rheumatoid arthritis, non-rheumatoid arthritidies, non-rheumatoid factor positive arthritis, ankylosing spondylitis, Lyme disease, and Reiter's syndrome; immune-mediated disorders such as glomerulonephritis, hemolytic uraemic syndrome, juvenile diabetes mellitus, mixed cryoglobulinaemia, polyarteritis, familial Mediterranean fever, amyloidosis, scleroderma, systemic lupus erythematosus, and Behgets syndrome; autoimmune disorders including systemic lupus, idiopathic thrombocytopenic purpura, Sjogren's syndrome, hemolytic uremic syndrome, and scleroderma; neurological syndromes such as chronic fatigue syndrome, migraine, multiple sclerosis, amyotrophic lateral sclerosis, myasthenia gravis, Gillain-Barre syndrome, Parkinson's disease, Alzheimer's disease, Chronic Inflammatory Demyelinating Polyneuropathy, and other degenerative disorders; psychiatric disorders including chronic depression, schizophrenia, psychotic disorders, manic depressive illness; regressive disorders including, Asperger's syndrome, Rett syndrome, attention deficit hyperactivity disorder (ADHD), and attention deficit disorder (ADD); the regressive disorder; autism; sudden infant death syndrome (SIDS); anorexia nervosa; dermatological conditions such as chronic urticaria, acne, dermatitis herpetiformis and vasculitis disorders; and cardiovascular and/or vascular disorders and diseases.

In various embodiments, the drug products of the present invention are used to treat or prevent a CDI or a C. difficile-associated disease. In various embodiments, the CDI or C. difficile-associated disease is one or more of: C. difficile diarrhea (CDD), C. difficile intestinal inflammatory disease, colitis, pseudomembranous colitis, fever, abdominal pain, dehydration and disturbances in electrolytes, megacolon, peritonitis, and perforation, and/or rupture of the colon. In various embodiments, the drug products of the present invention treat or prevent recurrent Clostridium difficile infection. In some embodiments, the drug products of the present invention are used to treat or prevent a diarrheal disease including, but not limited to, acute bloody diarrhea (e.g., dysentery), acute watery diarrhea (e.g., cholera), checkpoint inhibitor associated colitis, diarrhea due to food poisoning, persistent diarrhea, and traveler's diarrhea.

In some embodiments, the drug products of the present invention are used to treat or prevent an IBD or related disease including, but not limited to, Behcet's disease, collagenous colitis, Crohn's disease, diversion colitis, fulminant colitis, intermediate colitis, left-sided colitis, lymphocytic colitis, pancolitis, pouchitis, proctosigmoiditis, short bowel syndrome, ulcerative colitis, and ulcerative proctitis.

In various embodiments, the drug products of the present invention are used to treat or prevent the various Gl disorders disclosed herein and/or as known in the art to be a result of gut dysbiosis.

In some embodiments the mixtures of bacterial strains reduce Gl immunoactivation and inflammation.

In various embodiments, the drug products of the present invention are used to stimulate and/or activate Toll-like receptor activity (e.g., TLR1 , and/or TLR2, and/or TLR3, and/or TLR4, and/or TLR5, and/or TLR6, and/or TLR7, and/or TLR8, and/or TLR9, and/or TLR10, and/or TLR1 1 , and/or TLR12, and/or TLR13).

In various embodiments, the drug products of the present invention are used to treat or prevent various bloodstream infections (BSI).

In various embodiments, the drug products of the present invention are used to treat or prevent catheter or intravascular-line infections (e.g., central-line infections).

In various embodiments, the drug products of the present invention are used to treat or prevent chronic inflammatory diseases.

In various embodiments, the drug products of the present invention are used to treat or prevent meningitis.

In various embodiments, the drug products of the present invention are used to treat or prevent pneumonia, e.g., ventilator-associated pneumonia.

In various embodiments, the drug products of the present invention are used to treat or prevent skin and soft tissue infections.

In various embodiments, the drug products of the present invention are used to treat or prevent surgical-site infections. In various embodiments, the drug products of the present invention are used to treat or prevent urinary tract infections (e.g., antibiotic-resistant urinary tract infections and catheter-associated urinary tract infections).

In various embodiments, the drug products of the present invention are used to treat or prevent wound infections

In various embodiments, the drug products of the present invention are used to treat or prevent other well-known infections: antibiotic-resistant infections and antibiotic-sensitive infections.

Definitions

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

As used herein, the term "article" refers to a capsule of the present invention, e.g., comprising an inner layer coating and/or an outer layer coating. The term "pharmaceutical composition" is also used herein to refer to the capsule of the present invention, e.g., comprising an inner layer coating and/or an outer layer coating.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements).

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives [i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example, within plus or minus 10%.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements).

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 21 11 .03.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The alkyl groups may be optionally substituted, as described more fully below. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, 2-ethylhexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. "Heteroalkyl" groups are alkyl groups wherein at least one atom is a heteroatom (e.g., oxygen, sulfur, nitrogen, phosphorus.), with the remainder of the atoms being carbon atoms. Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl.

The term "alkoxy" refers to an alkyl group, as defined above, having an oxygen atom attached thereto. Representative alkoxy groups include methoxy, ethoxy, propyloxy, and tert-butoxy. An "ether" is two hydrocarbons covalently linked by an oxygen.

The term "substituted" is contemplated to include all permissible substituents of organic compounds, "permissible" being in the context of the chemical rules of valence known to those of ordinary skill in the art. In some cases, "substituted" may generally refer to replacement of a hydrogen with a substituent as described herein. However, "substituted," as used herein, does not encompass replacement and/or alteration of a key functional group by which a molecule is identified, e.g., such that the "substituted" functional group becomes, through substitution, a different functional group. For example, a "substituted phenyl" must still comprise the phenyl moiety and cannot be modified by substitution, in this definition, to become, e.g., a heteroaryl group such as pyridine. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic 1 0 compou nds .

Examples of substituents include, but are not limited to, alkyl, aryl, aralkyl, cyclic alkyl, heterocycloalkyl, hydroxy, alkoxy, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, amino, halogen, alkylthio, oxo, acyl, acylalkyl, carboxy esters, carboxyl, carboxamido, nitro, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like.

The term "aryl" refers to an aromatic carbocyclic group having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fused rings in which at least one is aromatic (e.g., 1 ,2,3,4- tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl), all optionally substituted. "Heteroaryl" groups are aryl groups wherein at least one ring atom in the aromatic ring is a heteroatom, with the remainder of the ring atoms being carbon atoms. Examples of heteroaryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N lower alkyl pyrrolyl, pyridyl N oxide, pyrimidyl, pyrazinyl, imidazolyl, indolyl and the like, all optionally substituted.

The terms "amine" and "amino" refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: N(R')(R")(R"') wherein R', R", and R'" each independently represent a group permitted by the rules of valence.

The terms "acyl," "carboxyl group," or "carbonyl group" are recognized in the art and can include such moieties as can be represented by the general formula:

Hv ,

wherein W is H, OH, O-alkyl, O-alkenyl, or a salt thereof. Where W is O-alkyl, the formula represents an "ester." Where W is OH, the formula represents a "carboxylic acid." In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where W is a S-alkyl, the formula represents a "thiolester." Where W is SH, the formula represents a "thiolcarboxylic acid." On the other hand, where W is alkyl, the above formula represents a "ketone" group. Where W is hydrogen, the above formula represents an "aldehyde" group.

The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur atom attached thereto. In some embodiments, the "alkylthio" moiety is represented by one of—S-alkyl,— S-alkenyl, and— S-alkynyl. Representative alkylthio groups include methylthio and ethylthio.

The term "amido" is art-recognized as an amino substituted by a carbonyl group .

The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group. The term "heteroaralkyl", as used herein, refers to an alkyl group substituted with a heteroaryl group.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Examplary heteroatoms are nitrogen, oxygen, and sulfur. As used herein, the term "thiol" means— SH; the term "hydroxyl" means— OH; and the term "sulfonyl" means— S02— .

As used herein the term "oxo" refers to a carbonyl oxygen atom.

Any terms as used herein related to shape and/or geometric relationship of or between, for example, one or more articles, structures, and/or subcomponents thereof and/or combinations thereof and/or any other tangible or intangible elements not listed above amenable to characterization by such terms, unless otherwise defined or indicated, shall be understood to not require absolute conformance to a mathematical definition of such term, but, rather, shall be understood to indicate conformance to the mathematical definition of such term to the extent possible for the subject matter so characterized as would be understood by one skilled in the art most closely related to such subject matter. Examples of such terms related to shape and/or geometric relationship include, but are not limited to terms descriptive of: shape, such as, round, square, circular/circle, rectangular/rectangle, triangular/triangle, cylindrical/cylinder, elliptical/ellipse, (n)polygonal/(n)polygon.; surface and/or bulk material properties and/or spatial/temporal resolution and/or distribution - such as, smooth, reflective, transparent, clear, opaque, rigid, impermeable, uniform(ly), inert, non-wettable, insoluble, steady, invariant, constant, homogeneous.; as well as many others that would be apparent to those skilled in the relevant arts. As one example, a fabricated article that would described herein as being " square" would not require such article to have faces or sides that are perfectly planar or linear and that intersect at angles of exactly 90 degrees (indeed, such an article can only exist as a mathematical abstraction), but rather, the shape of such article should be interpreted as approximating a " square," as defined mathematically, to an extent typically achievable and achieved for the recited fabrication technique as would be understood by those skilled in the art or as specifically described.

Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

This invention is further illustrated by the following non-limiting example.

EXAMPLE

In this non-limiting example, multiple concentrations of shellac and ethanol were evaluated to identify a possibly optimal solution ratio of a hydrophobic coating for an inner layer.

Gelatin capsules were provided an outer Phloral® coating. Here, a solution comprising 56% EUDRAGIT® S100 (poly(methacrylic acid, methylmethacrylate)), 24% Starch, 12% Triethyl Citrate, and 8% PlasACRYL T20 dissolved in a solution of water, ethanol, and n-butanol was prepared and then charged to a Fluid Bed Coater. The solution was then spray coated on the outer surface of the capsule bodies and capsule caps to a target weight gain. The capsule bodies and capsule caps were allowed to dry for about 8 hours to about 24 hours before further procession.

Concentrations of 0.1 g shellac in 1 ml ethanol (200 proof) to 1 .3 g shellac to 1 ml ethanol were coated on the interior of gelatin/Phloral® outer coated capsules.

In this example, using a size 00 capsule, 900 μΙ to 1000 μΙ of a hydrophobic coating was added to each capsule body and 125 μΙ to 200 μΙ of a hydrophobic coating was added to each capsule cap. As mentioned above, the caps were not entirety filled with the hydrophobic coating. Thus, there was a ring around the internal surface of the open end of the capsule cap which did not comprise an inner coating layer. This ring allowed a gentle seal to form between capsule cap and the capsule body. Absent the ring, the excess material on the open end of the capsule may have prevented sealing of the capsule cap onto the capsule body or it may have required additional force to enact a seal.

Soon after filling, excess coating was removed and the capsule bodies and capsule caps were allowed to dry, for at least 24 hours to 48 hours.

A dissolution assay, which determined the point of aqueous phase release within a simulated gastrointestinal tract, was utilized to evaluate the effectiveness of the formulations.

The dissolution assay included the following steps (multiple capsule types were run through the assay in parallel). First, a capsule was placed in simulated gastric fluid (SGF) with pepsin at pH 1 .2 according to USP and agitated vigorously for approximately three hours, thereby mimicking residence in the stomach. Then, the SGF is replaced with simulated intestinal fluid (SIF) with pancreatin at pH 6.8 according to USP; the capsule was lightly agitated for approximately two hours, thereby mimicking transit through the small intestine. Finally, the pH of the SIF was adjusted to 7.4; the capsules were lightly agitated for approximately two hours, thereby mimicking passage into the colon. Since the delayed-release capsules of the present invention are designed for enteric release, mechanical disintegration and detectable levels of aqueous phase (e.g., drug product) release should have occurred about two hours after the SIF was adjusted to pH 7.4, when conditions mimic passage to the upper colon. The dissolution assay compared gelatin/Phloral® outer coated capsule capsules filled with a drug product (frozen stool) and lacking an inner coating and similarly filed gelatin/Phloral® outer coated capsules which comprise an inner layer.

Here, the, delayed-release capsules were filled with an aqueous phase comprising a drug product or with an aqueous phase having a pH, osmolality, and/or viscosity equivalent to a desired drug product (e.g., an aqueous phase comprising glycerol) and comprising a detectable substance, e.g., food coloring.

Capsule performance was denoted as pass or fail. Pass was defined as release within the simulated large intestine. Fail was defined as release within the simulated stomach. It was determined that concentrations of 0.7, 0.8, 0.9, and 1.0 g shellac per 1 ml of ethanol provided good results; with the 1 gram of shellac to 1 mL of ethanol [i.e., 1 :1 ratio), found to be the best performing of the tested conditions. See, below Table 1 .

Table 1

Coated capsules were weighed to determine the target weight gain to provide adequate quality and release profile as tested in a dissolution assay. The following weight ranges were found to be desirable: bodies 0.4 g to 0.9 g and caps: 0.01 g to 0.03 g.

Finally, dissolution assays were performed which compared gelatin capsules filled with a drug product (frozen stool) and lacking an inner coating and similarly filed capsules which comprise an inner layer formed from 1 :1 ratio of shellac to ethanol hydrophobic coating. Data obtained is shown in Table 2:

Table 2

On average, the capsules lacking an inner layer began to release the drug product five minutes after being placed in the SGF; complete depletion of the drug product was observed by thirty minutes. In contrast, on average, only 15% of the inner coated capsules of the present invention released drug product after three hours in the SGF environment; the remaining 85% of the inner coated capsules of the present invention released their drug product in the SIF environment.

Claims

CLAIMS What is claimed is:

1 . A pharmaceutical composition for administration of a microbial composition in an aqueous phase, comprising:

a capsule including a capsule body and a capsule cap comprising a degradable material, the capsule having an inner surface and an outer surface, wherein an inner layer comprising a hydrophobic material coats the inner surface and an outer layer comprising a delayed release material coats the outer surface; and

an aqueous phase comprising the microbial composition encapsulated by the capsule, wherein the inner layer inhibits or substantially reduces dissolution of the capsule.

2. The pharmaceutical composition of claim 1 , wherein the degradable material at least partially degrades or dissolves in the presence of an aqueous phase at ambient conditions.

3. The pharmaceutical composition of any one of claims 1 or 2, wherein the degradable material is selected from the group consisting of gelatin and a derivative thereof, HPMC, alginate and a derivative thereof, polylactic acid, polyglycolic acid, copolymers thereof, and a combination thereof.

4. The pharmaceutical composition of any one of the preceding claims, wherein the degradable material is gelatin.

5. The pharmaceutical composition of any one of the preceding claims, wherein a portion of the capsule would degrade, dissolve, leak, and/or crack if contacted with the aqueous phase. 6. The pharmaceutical composition of any one of the preceding claims, wherein the portion of the capsule would degrade, dissolve, leak, and/or crack less than 1 day, 12 hours,

6 hours, 3 hours, or 1 hour after contacting the aqueous phase.

7. The pharmaceutical composition of any one of the preceding claims, wherein the capsule further comprises an enteric polymer.

8. The pharmaceutical composition of any one of the preceding claims, wherein the enteric polymer comprises a EUDRAGIT® (poly(meth)acrylate).

9. The pharmaceutical composition of any one of the preceding claims, wherein the inner layer is disposed between the aqueous phase and the inner surface of the capsule.

10. The pharmaceutical composition of any one of the preceding claims, wherein the inner layer covers substantially the entire inner surface of the capsule body.

1 1 . The pharmaceutical composition of any one of the preceding claims, wherein the inner layer covers the entire inner surface of the capsule body.

12. The pharmaceutical composition of any one of the preceding claims, wherein the inner layer does not cover a portion of the inner surface of the capsule cap.

13. The pharmaceutical composition of any one of the preceding claims, wherein the portion of the inner surface of the capsule cap that is not covered by the inner layer is located at the open end of the cap.

14. The pharmaceutical composition of any one of the preceding claims, wherein the portion of the inner surface of the capsule cap that is not covered by the inner layer includes the portion of the capsule cap that overlaps the capsule body when the capsule is sealed.

15. The pharmaceutical composition of any one of the preceding claims, wherein the hydrophobic material of the inner layer is selected from the group consisting of zein, polysaccharides, silk, polycaprolactone, oil, pectin, wax, polymers, shellac and a derivative thereof, and a combination thereof.

16. The pharmaceutical composition of any one of the preceding claims, wherein the hydrophobic material of the inner layer is shellac.

17. The pharmaceutical composition of any one of the preceding claims, wherein the inner layer further comprises an enteric polymer.

18. The pharmaceutical composition of any one of the preceding claims, wherein the enteric polymer comprises a EUDRAGIT® (poly(meth)acrylate).

19. The pharmaceutical composition of any one of the preceding claims, wherein the inner layer prevents or inhibits the aqueous phase from contacting the degradable material of the capsule.

20. The pharmaceutical composition of any one of the preceding claims, wherein the inner layer prevents or inhibits the aqueous phase from degrading, dissolving, and/or cracking the capsule and/or causing the capsule to leak.

21 . The pharmaceutical composition of any one of the preceding claims, wherein a portion of the capsule lacking an inner layer would degrade, dissolve, leak, and/or crack less than 1 day, 12 hours, 6 hours, 3 hours, or 1 hour after contacting the aqueous phase.

22. The pharmaceutical composition of any one of the preceding claims, wherein the outer layer covers substantially the entire outer surface of the capsule.

23. The pharmaceutical composition of any one of the preceding claims, wherein the outer layer covers the entire outer surface of the capsule.

24. The pharmaceutical composition of any one of the preceding claims, wherein the delayed release material comprises an enteric polymer.

25. The pharmaceutical composition of any one of the preceding claims, wherein the enteric polymer comprises a EUDRAGIT® (poly(meth)acrylate).

26. The pharmaceutical composition of any one of the preceding claims, wherein the enteric polymer comprises Phloral®.

27. The pharmaceutical composition of any one of the preceding claims, wherein the outer layer prevents, inhibits, or delays a moist or aqueous external environment from contacting the degradable material of the capsule.

28. The pharmaceutical composition of any one of the preceding claims, wherein the outer layer prevents, inhibits, or delays the moist or aqueous external environment from degrading, dissolving, and/or cracking the capsule and/or causing the capsule to leak.

29. The pharmaceutical composition of any one of the preceding claims, wherein a portion of the capsule lacking an outer layer would degrade, dissolve, leak, and/or crack less than 12 hours, 6 hours, 4 hours, 2 hours, or 1 hour after contacting the moist or aqueous external environment.

30. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition is stable at room temperature for at least 1 day, 7 days, 1 month, or 1 year.

31 . The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition is stable at 37 °C at a pH less than 7, pH less than 6, a pH less than 5 a pH less than 4, a pH less than 3, or a pH less than 2 for at least 1 hour.

32. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition begins to dissolve, degrade, crack, and/or leak in less than 6 hours, 4 hours, 2 hours, or 1 hour at 37 °C at a pH less greater than 7.

33. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition begins to dissolve, degrade, crack, and/or leak in less than 4 hours, 2 hours, at 1 hour at

37 °C at a pH less greater than 7.

34. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition begins to dissolve, degrade, crack, and/or leak in less than 6 hours, 4 hours, 2 hours, or 1 hour at 37 °C at a pH less greater than 8.

35. The pharmaceutical composition of any one of the preceding claims, wherein the microbial composition is human stool or a derivative thereof.

36. The pharmaceutical composition of any one of the preceding claims, wherein the microbial composition comprises bacteria, fungi, phages, viruses, fiber, and/or mucus.

37. The pharmaceutical composition of any one of the preceding claims, wherein the microbial composition comprises viable bacteria.

38. The pharmaceutical composition of any one of the preceding claims, wherein the capsule, the inner layer, the outer layer, and/or the aqueous phase comprises an additive.

39. The pharmaceutical composition of any one of the preceding claims, wherein the additive is selected from the group consisting of starches, genipin, aloe, whey protein, guar gum, xantham gum, and carrageenan.

40. A method for delivering a microbial composition to the gastrointestinal (Gl) tract of a subject, comprising a step of administering a pharmaceutical composition of any of the above claims to the subject.

41 . The method of claim 40, wherein the administering is oral administration.

42. The method of claim 40 or claim 41 , wherein the Gl tract comprises one or more of the duodenum, small intestine, duodenum, jejunum, ileum, large intestine, colon transversum, colon descendens, colon ascendens, colon sigmoidenum, cecum, and rectum.

43. The method of claim 42, wherein the large intestine comprises one or more of colon transversum, colon descendens, colon ascendens, colon sigmoidenum, and cecum.

44. The method of claim 42, wherein the small intestine comprises one or more of duodenum, jejunum, and ileum.

45. The method of any one of the claims 40 to 44, wherein the subject has a disease/disorder associated with the presence of abnormal enteric microflora.

46. A method for forming a delayed-release capsule, comprising:

forming an outer layer which coats at least a portion of an outer surface of a capsule body and forming an outer layer which coats at least a portion of the outer surface of a capsule cap with a solution comprising a delayed release material; and

forming an inner layer which coats at least a portion of an inner surface of a capsule body and forming an inner layer which coats at least a portion of the inner surface of a capsule cap with a solution comprising a hydrophobic material.

47. The method of claim 46, wherein the step of forming an outer layer precedes the step of forming an inner layer.

48. The method of claim 46 or claim 47, wherein the solution comprising a delayed release material is prepared by dispersing one or more enteric polymers in a solution.

46. The method of any one of the claims 46 to 48, wherein the step of forming an outer layer comprises spraying the solution comprising a delayed release material onto the outer surface of a capsule body and/or onto the outer surface of a capsule cap.

50. The method of any one of the claims 46 to 49, wherein the outer layer covers substantially the entire outer surface of the capsule body and the capsule cap.

51 . The method of any one of the claims 46 to 50, wherein the outer layer covers the entire outer surface of the capsule body and the capsule cap.

52. The method of any one of the claims 46 to 51 , wherein the enteric polymer comprises a EUDRAGIT® (poly(meth)acrylate).

53. The method of any one of the claims 46 to 52, wherein the solution comprising a delayed release material comprises at least 50% EUDRAGIT® (poly(meth)acrylate).

54. The method of any one of the claims 46 to 53, wherein the enteric polymer comprises Phloral®.

55. The method of any one of the claims 46 to 54, wherein the outer layer is dried for between about 8 hours and about 24 hours.

56. The method of any one of the claims 46 to 55, wherein the solution comprising a hydrophobic material is prepared by dispersing one or more hydrophobic materials in a solution.

57. The method of any one of the claims 46 to 56, wherein the solution comprising a hydrophobic material comprises an alcohol.

58. The method of any one of the claims 46 to 57, wherein the alcohol is ethanol.

59. The method of any one of the claims 46 to 58, wherein the ethanol is 200-proof ethanol.

60. The method of any one of the claims 46 to 59, wherein the hydrophobic material is selected from the group consisting of zein, polysaccharides, silk, polycaprolactone, oil, pectin, wax, polymers, shellac and a derivative thereof, and a combination thereof.

61 . The method of any one of the claims 46 to 60, wherein the hydrophobic material is shellac.

62. The method of any one of the claims 46 to 61 , wherein the solution comprising a hydrophobic material comprises between about 0.1 g shellac per ml ethanol to about 1 .3 g shellac per ml of ethanol

63. The method of any one of the claims 46 to 62, wherein the solution comprising a hydrophobic material comprises between about 0.7, 0.8, 0.9, and 1 .0 g shellac per ml ethanol.

64. The method of any one of the claims 46 to 63, wherein the solution comprising a hydrophobic material comprises a 1 :1 ratio of shellac (in grams) to ml of ethanol.

65. The method of any one of the claims 46 to 64, wherein the step of forming an inner layer on at least a portion of an inner surface of a capsule body comprises substantially filling the capsule body with the solution comprising a hydrophobic material, removing the solution comprising the hydrophobic material, and drying the capsule body for about 24 hours to about three days.

66. The method of any one of the claims 46 to 65, wherein the step of forming an inner layer on at least a portion of an inner surface of a capsule cap comprises filling the capsule cap up to a defined level of the capsule cap with the solution comprising a hydrophobic material, removing the solution comprising a hydrophobic material, and drying the capsule cap for 24 hours to three days.

67. The method of any one of the claims 46 to 66, wherein the defined level of the capsule cap excludes the portion of the capsule cap that overlaps the capsule body when the capsule is sealed.

68. The method of any one of the claims 46 to 67, wherein when the capsule is a size 00 capsule, between about 50 μΙ and about 400 μΙ of the hydrophobic coating solution of the hydrophobic coating solution is dispensed per capsule cap.

69. The method of any one of the claims 46 to 68, wherein when the capsule is a size 00 capsule, a capsule body comprising an inner layer weighs between about 0.4 g and about 0.9 g and a capsule cap comprising an inner layer weighs between about 0.01 g and about 0.03 g.

70. The method of any one of the claims 46 to 69, wherein the capsule comprises a degradable material that is selected from the group consisting of gelatin and a derivative thereof, HPMC, alginate and a derivative thereof, polylactic acid, polyglycolic acid, copolymers thereof, and a combination thereof.

71 . The method of any one of the claims 46 to 70, wherein the degradable material is gelatin.

72. A method for forming a capsule filled with microbial composition in an aqueous phase, comprising:

obtaining the delayed-release capsule obtained by the method of any of claims 46 to

71 ,

filling the capsule body with the microbial composition in an aqueous phase, and sealing the capsule body with a capsule cap.

73. The method of claim 72, wherein the microbial composition is human stool or a derivative thereof.

74. The method of claim 72 or claim 73, wherein the microbial composition comprises bacteria, fungi, phages, viruses, fiber, and/or mucus.

75. The method of any one of claims 72 to 74, the microbial composition comprises viable bacteria.