Long-term stable live fecal microbiota composition

Abstract

The present invention provides a solid oral pharmaceutical composition comprising a pharmaceutically effective amount of live microorganisms and one or more pharmaceutically acceptable water-absorbing excipients, wherein the water content of the composition is between 0.5% and 30% by weight of the total composition, as determined according to the European Pharmacopoeia 9.4 section 2.5.12. The present invention also provides a process for its preparation and its use in therapy. The live cell-based composition of the present invention is stable under mild conditions.

Description

[0001] This application claims the benefit of European patent application EP19382287.1, filed on April 15, 2019.

[0002] This invention relates to compositions containing a stable community of live fecal microorganisms. Background Technology

[0003] Fecal microbiota transplantation (FMT) is the transfer of fecal material containing microorganisms from a healthy individual to a diseased recipient.

[0004] Traditionally, transplantation to the upper gastrointestinal (GI) tract is achieved via nasogastric tube, nasoduodenal tube, nasojejunal tube, or through esophagogastric endoscopy or push-through enteroscopy. Delivery to the lower gastrointestinal tract is typically via colonoscopy, sigmoidoscopy, or an enema. All of these techniques have drawbacks. For example, upper gastrointestinal delivery carries the risk of aspiration-related complications (especially with nasogastric delivery) and is invasive and uncomfortable for the recipient. Lower gastrointestinal delivery techniques such as colonoscopy and sigmoidoscopy are also invasive and uncomfortable, and are associated with significant costs and risks.

[0005] Therefore, there is still a need for a safe, effective, and less invasive method to deliver microbial communities to recipients (e.g., fecal transplantation or fecal microbiota transplantation).

[0006] Two methods have been used to develop encapsulated oral formulations of microbial communities: (a) rapid freezing of fecal aqueous solutions; and (b) freeze-drying.

[0007] The first method involves rapidly freezing an aqueous solution of feces in a glycerol and saline buffer. The aqueous solution preserves the viability of the microbial strains but produces highly unstable capsules because the water-containing properties of feces rapidly degrade the water-soluble capsules. The physical instability of these capsules complicates mass production and poses clinical hazards, as the capsules can rupture during administration. This instability necessitates that the capsules be stored at -70°C / -80°C before their use (i.e., the moment they must be thawed). This instability, along with the need for "complex" equipment, limits fecal microbiota therapy (FMT) to hospital settings under special medical supervision. Therefore, patients seeking treatment must go to hospitals equipped with all the appropriate means to freeze and thaw the capsules. However, this leads to a lack of treatment adherence, resulting in patient non-compliance.

[0008] Several attempts have been made to reduce the water content of feces. Dehydration of the microbial community using techniques such as freeze-drying is one of the main approaches. However, the dehydration process is physically demanding and significantly reduces the viability of the microorganisms. Furthermore, sometimes the dehydration of the microbial community is insufficient to achieve adequate stability, thus necessitating the development of specialized containers. For example, KR20080059605 discloses packaging freeze-dried bacteria in a specially designed container to reduce the humidity of the environment in contact with the capsule. This Korean patent document provides stability data for freeze-dried bacteria containing (Example 6) or without (Example 5) excipients. Example 6 provides data for capsules containing freeze-dried bacteria, microcrystalline cellulose, and magnesium stearate. Notably, capsules without excipients are more stable in the specific container than when the capsules are formulated with excipients.

[0009] Despite the efforts made to date, a stable, home-use FMT based on live bacterial cells is still needed. Summary of the Invention

[0010] The inventors have developed a live microbiome capsule that is stable enough to be stored at about 4°C rather than -65°C or -80°C, meaning that the capsule can be stored in a regular refrigerator in the recipient's home.

[0011] As shown below, the inventors first prepared a mixture of fecal microbiota (obtained from an aqueous solution of feces in glycerol) with microcrystalline cellulose (hereinafter also referred to as "MCC") and a lubricant (magnesium stearate). When capsules were prepared using this mixture, according to European Pharmacopoeia 9.4, section 2.5.12, it was found that their moisture content accounted for approximately 30% of the total weight of the composition (see Table 1 below).

[0012] Surprisingly, the inventors have discovered that the capsules containing live microbiota in environments with a moisture content as high as approximately 30% remain stable for 3 months at 4°C (equivalent to a refrigerator temperature), even at such high moisture levels. In this regard, Table 1 shows that the addition of a water-absorbing excipient imparts stability to the encapsulated live microbiota, a stability on the same order of magnitude as that achieved by freeze-dried microbiota. Therefore, the addition of a water-absorbing excipient is an effective alternative to freeze-drying technology (which is more "aggressive" to bacterial activity).

[0013] This is unexpected from the perspective of existing technology. As mentioned above, existing technology has taught two options for achieving stable encapsulated fecal microbiota: lowering the temperature to approximately -80°C to obtain frozen fecal microbiota; or reducing the moisture content as much as possible by freeze-drying the fecal microbiota. Even recent publications have disclosed the encapsulation of freeze-dried bacteria and the packaging of the resulting freeze-dried bacteria in special containers that further reduce the moisture content within the container (e.g., Korean Patent KR20080059605). Therefore, the present invention, which provides a live fecal microbiota stable at a milder temperature (requiring a higher moisture content compared to freeze-drying), is surprising from the perspective of existing technology.

[0014] The stability of capsules is reflected not only in bacterial activity but also in capsule morphology. As described below, when capsules formulated with fecal microbiota and water-absorbing excipients (MCC) were stored at 4°C for three months, no changes in length, width, or odor were observed.

[0015] Therefore, in summary, the compositions of the present invention represent a significant advancement in the field of FMT, as recipients can undergo treatment at home without having to visit a medical clinic. Furthermore, the compositions of the present invention are advantageous because they can improve treatment adherence by providing enhanced convenience to patients, thereby leading to increased patient persistence.

[0016] Therefore, in a first aspect, the present invention provides a solid oral pharmaceutical composition comprising a pharmaceutically effective amount of live microorganisms and one or more pharmaceutically acceptable water-absorbing excipients, wherein the water content of the mixture is from 0.5% to 30% of the total weight of the composition, as determined according to Section 2.5.12. of the European Pharmacopoeia 9.4.

[0017] In a second aspect, the present invention provides a method for preparing an oral pharmaceutical composition as defined in the first aspect of the present invention, the method comprising mixing live microorganisms with one or more water-absorbing excipients.

[0018] Compared with the prior art, the advantage of the present invention is that the method for obtaining the composition of the present invention requires very few steps under mild conditions (room temperature and humidity), which minimizes the risk of loss of live bacteria.

[0019] In a third aspect, the present invention provides an oral pharmaceutical composition that can be obtained by the method defined in the second aspect of the present invention.

[0020] In a fourth aspect, the present invention provides oral pharmaceutical compositions as defined in the first or third aspect of the invention for therapeutic purposes.

[0021] This is the first report on the ability of water-absorbing excipients to stabilize encapsulated live microorganisms. In fact, based on the data provided in Korean Patent KR20080059605, when comparing the data provided in Examples 5 and 6 of the prior art, those skilled in the art would expect that water-absorbing excipients would have a negative impact on the stability of the microbiome.

[0022] Unbound by theory, the inventors believe this surprising effect is due to the interaction between the water-absorbing excipient (e.g., MCC, as shown below) and the water present in the fecal microbiota, where some water remains in a "free" state while a small amount is absorbed as "structured water," resulting in the formation of a molecular sponge. In this way, the solid pharmaceutical composition will contain up to approximately 30% water (which would correspond to the water content of the initial fecal microbiota used to prepare the capsule), but most of the water will be contained within a formed "sponge" (the physical form taken by the absorbent excipient). This "sponge" can act as a reservoir or protective barrier: under specific environmental conditions, the living microbiota within the capsule can gradually utilize the available water within the capsule. Thus, the water-absorbing excipient is able to exert a stabilizing effect due to the water content in the mixture.

[0023] The above explains why the same water-absorbing excipient (MCC) in Korean Patent 20080059605 cannot provide this stabilizing effect: the excipient cannot stabilize microorganisms because it cannot absorb enough water because: (a) the bacteria are freeze-dried (i.e., do not contain a large amount of water); and (b) the ambient humidity is low due to the special design of the container.

[0024] Therefore, in a fifth aspect, the present invention provides the use of water-absorbing excipients for stabilizing live microorganisms in solid oral pharmaceutical compositions.

[0025] Finally, the present invention also provides solid oral compositions as defined in the first or third aspect of the invention for use in the treatment of dysbiosis-related diseases. This aspect can alternatively be expressed as the use of the solid oral compositions as defined in the first or third aspect of the invention in the preparation of a medicament for treating dysbiosis-related diseases. This aspect can alternatively be expressed as a method of treating or preventing dysbiosis-related diseases, the method comprising administering a therapeutically effective amount of the composition as defined in the first or third aspect of the invention to a subject in need. Detailed Implementation

[0026] Unless otherwise stated, all terms used herein should be understood in their ordinary meaning as known in the art. Unless otherwise expressly listed, more specific definitions of certain terms used herein are described below and are intended to be applied uniformly throughout the specification and claims. Furthermore, for the purposes of this invention, any range given includes both the lower and upper endpoints of that range. Unless otherwise specified, given ranges, such as temperature, time, weight, etc., should be considered approximate.

[0027] As described above, in a first aspect, the present invention relates to oral solid pharmaceutical compositions comprising live microorganisms and one or more water-absorbing excipients.

[0028] As used herein, the term "therapeutic effective amount" refers to an amount of live microorganisms that, when applied, is sufficient to prevent the development of one or more symptoms of the disease to be addressed, or to alleviate, to some extent, one or more symptoms of the disease to be addressed. Of course, the specific dosage of live microorganisms applied according to the present invention will depend on the specific circumstances of the case, including the compound applied, the route of application, the specific disease being treated, and similar considerations.

[0029] The term "pharmaceutical composition" refers to those compositions that have beneficial effects on both humans and non-humans.

[0030] In one embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided below, wherein the live microorganisms are probiotic microorganisms or fecal microbiota. In another embodiment, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the live microorganisms are fecal microbiota.

[0031] In the context of this paper, the term "microbiota" refers to the community of microorganisms present (persistently or transiently) in and on animal objects, typically mammals such as humans, including eukaryotes, archaea, bacteria, fungi such as yeast, and viruses (including bacterial viruses, i.e., bacteriophages). Fecal microbiota includes a large number of unknown but diverse types of microorganisms.

[0032] In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition comprising a water-absorbing excipient.

[0033] In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the water-absorbing excipient is selected from: cellulose-based excipients or pharmaceutically acceptable salts thereof; kaolinite; talc; palygorskite; sepiolite; colloidal silica; and smectites (of which montmorillonite, saponite, and lithium montmorillonite are the most widely used). In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the water-absorbing excipient is a cellulose-based excipient. In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the water-absorbing excipient is a cellulose ether derivative (e.g., alkyl (e.g., C...). 1-10 Alkyl ethers, hydroxyalkyl ethers (e.g., HO-(C 1-10 alkyl ethers, or carboxyl ethers (e.g., OH(O)C-(C) 1-10 Alkyl ethers, or pharmaceutically acceptable salts thereof; cellulose esters (e.g., cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate (CAP), cellulose acetate butyrate (CAB) derivatives) or mixtures thereof (i.e., mixtures of one or more ether derivatives with one or more ether derivatives, mixtures of one or more ether derivatives with one or more ester derivatives, or mixtures of one or more ester derivatives with one or more ester derivatives). In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the cellulose-based excipient is selected from: methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylethylcellulose, carboxymethylcellulose, and microcrystalline cellulose (MCC). In another embodiment, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the water-absorbing excipient is a cellulose ether derivative. In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the water-absorbing excipient is MCC. In another embodiment of the composition of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above, the composition comprising fecal microbiota and MCC.

[0034] As used herein, the term "pharmaceutically acceptable salt" means that salts suitable for contact with tissues of humans and lower animals, within reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Examples of pharmaceutically acceptable, non-toxic acid addition salts are amino salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (e.g., acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or by using other methods used in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include adipates, alginates, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, hydrogen sulfates, borates, butyrates, camphorates, camphor sulfonates, citrates, cyclopentanepropionates, disaccharides, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucono-enolates, glyceryl phosphates, gluconates, hemisulfates, heptarates, hexanoates, hydroiodates, 2-hydroxy-ethanesulfonates, lacturonates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, dihydroxynaphthalates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, neopentanoates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Salts derived from suitable bases include alkali metals, alkaline earth metals, and ammonium. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions (e.g., halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates).

[0035] The pharmaceutical composition of the present invention is characterized by a water content of up to 30%. In one embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the water content is 1% to 30%, 5% to 30%, or 9% to 30% of the total weight of the composition. Alternatively, the water content of the pharmaceutical composition of the first aspect of the invention may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the total weight of the composition.

[0036] In another embodiment of the first aspect of the invention, optionally combined with any of the embodiments provided above or below, the solid oral pharmaceutical composition comprises a water content of 1% to 30%, 5% to 30%, or 9% to 30% by weight of the total composition, and the water-absorbing excipient is selected from: cellulose-based excipients or pharmaceutically acceptable salts thereof; kaolinite; talc; palygorskite; sepiolite; colloidal silica; and montmorillonite (of which kaolinite, saponite, and lithium montmorillonite are the most widely used). In another embodiment of the first aspect of the invention, optionally combined with any of the embodiments provided above or below, the solid oral pharmaceutical composition comprises a water content of 1% to 30%, 5% to 30%, or 9% to 30% by weight of the total composition, and the water-absorbing excipient is a cellulose-based excipient. In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the solid oral pharmaceutical composition comprises a water content of 1% to 30%, 5% to 30%, or 9% to 30% by weight of the total composition, and the water-absorbing excipient is a cellulose ether derivative (e.g., alkyl (e.g., C...)). 1-10 Alkyl ethers, hydroxyalkyl ethers (e.g., HO-(C 1-10 alkyl ethers, or carboxyl ethers (e.g., OH(O)C-(C) 1-10Alkyl ethers, or pharmaceutically acceptable salts thereof; cellulose esters (e.g., cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate (CAP), cellulose acetate butyrate (CAB) derivatives) or mixtures thereof (i.e., mixtures of one or more ether derivatives with one or more ether derivatives, mixtures of one or more ether derivatives with one or more ester derivatives, or mixtures of one or more ester derivatives with one or more ester derivatives). In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the solid oral pharmaceutical composition comprises a water content of 1% to 30%, 5% to 30%, or 9% to 30% by weight of the total composition, and the cellulose-based excipient is selected from: methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylethylcellulose, carboxymethylcellulose, and microcrystalline cellulose (MCC). In one embodiment, optionally combined with any of the embodiments provided above or below, the solid oral pharmaceutical composition comprises a water content of 1% to 30%, 5% to 30%, or 9% to 30% by weight of the total composition, and the water-absorbing excipient is a cellulose ether derivative. In another embodiment of the first aspect of the invention, optionally combined with any of the embodiments provided above or below, the water-absorbing excipient is MCC. In another embodiment of the composition of the first aspect of the invention, optionally combined with any of the embodiments provided above, the solid oral pharmaceutical composition comprises a water content of 1% to 30%, 5% to 30%, or 9% to 30% by weight of the total composition, and further comprises fecal microbiota and MCC.

[0037] The water content was determined according to "European Pharmacopoeia 9.4 Section 2.5.12, page 5107 'Water: Semi-micro determination'", which is based on the reaction of water with sulfur dioxide and iodine in a suitable anhydrous medium in the presence of a base with sufficient buffering capacity. The measurement was performed using a device consisting of a titration vessel with two identical platinum electrodes.

[0038] In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition comprising one or more additional pharmaceutically or veterinary excipients.

[0039] The expression "pharmaceutically or veterinarily acceptable excipient or carrier" refers to a pharmaceutically acceptable material, composition, or solvent. Each component must be pharmaceutically acceptable in the sense of compatibility with other components of the pharmaceutical composition. It must also be suitable for contact with human and non-human animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio. Examples of suitable pharmaceutically acceptable excipients are lubricants, cryoprotectants, etc. Any conventional excipient medium is considered to be within the scope of this invention unless it is incompatible with a substance or its derivatives, for example, by producing any adverse biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition.

[0040] In one embodiment of the composition of the first aspect of the invention, the embodiment may optionally be combined with any of the embodiments provided above or below, the composition further comprising one or more pharmaceutically acceptable excipients selected from: cryoprotectants, lubricants, and combinations thereof.

[0041] The composition may contain at least one cryoprotectant. Examples of cryoprotectants that may be used are glycerol, carbohydrates, water-soluble antioxidants such as sodium ascorbate, glutathione, riboflavin, L-cysteine, and pharmaceutically acceptable salts thereof, or combinations thereof. In one embodiment of the composition of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the composition contains a cryoprotectant. In one embodiment of the composition of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the composition contains glycerol.

[0042] In one embodiment of the composition of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition comprising a cryoprotectant and a lubricant. In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition further comprising glycerin and a lubricant. In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition further comprising a cryoprotectant and a stearate. In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition further comprising glycerin and a stearate. In another embodiment of the first aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition further comprising glycerin and magnesium stearate.

[0043] In another embodiment of the first aspect of the present invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition comprising: fecal microbiota, cellulose derivative, stearate, and cryoprotectant. In another embodiment of the first aspect of the present invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition comprising: fecal microbiota, cellulose ether derivative, stearate, and glycerol. In another embodiment of the first aspect of the present invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition comprising: fecal microbiota, MCC, stearate, and glycerol. In another embodiment of the first aspect of the present invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition comprising: fecal microbiota, MCC, stearate, and glycerol. In another embodiment of the first aspect of the present invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition comprising: fecal microbiota, cellulose ether derivative, magnesium stearate, and glycerol. In another embodiment of the first aspect of the present invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the composition comprising: fecal microbiota, MCC, magnesium stearate, and glycerol.

[0044] In another embodiment of the first aspect of the invention, optionally combined with any of the embodiments provided above or below, the oral solid pharmaceutical composition is a capsule, i.e., a single capsule, such as a hard capsule or a soft capsule. In this invention, the expression "single capsule" means that the oral pharmaceutical composition consists of only one capsule containing a microbiome and an adsorbent. Therefore, this embodiment (i.e., "single capsule") does not include the possibility that the capsule containing the microbiome and the adsorbent is contained within another capsule. In another embodiment of the first aspect of the invention, optionally combined with any of the embodiments provided above or below, the solid oral pharmaceutical composition consists of a single capsule made of a microbiome and an adsorbent.

[0045] As used herein, the term "capsule" refers to a conventional hard capsule intended for oral administration to humans or animals. The capsules of the present invention are structurally consistent with the conventional definition of a hard capsule. When "capsule" is referred to herein, unless the context otherwise requires, it means an outer capsule or an inner capsule or an outer capsule containing an inner capsule. Generally, the term "capsule" refers to both empty and filled capsules, while "shell" specifically refers to an empty capsule.

[0046] As is known to those skilled in the art, commercially available capsules, whether ordinary or extended, are named by a number, and extended capsules have the suffix el.

[0047] Another advantage is that the production of the capsules of the present invention does not require drying, which would result in a significant loss of live bacteria: the mixture is simply filled into the capsules (using any conventional technique known to those skilled in the art).

[0048] In one embodiment of the first aspect of the invention, which may optionally be combined with any of the embodiments provided above or below, the composition is an enteric-coated capsule. The term "enteric-coated capsule" refers to such a capsule having enteric properties. "Enteric properties" means that the capsule is soluble in or decomposed by alkaline intestinal secretions, but substantially insoluble in or resistant to acidic gastric secretions. The mixtures provided by the present invention are also used to fill commercially available enteric-coated capsules.

[0049] All embodiments provided under the first aspect of the present invention are also embodiments of the second, third, fourth, and fifth aspects.

[0050] The present invention provides, in a second aspect, a method for preparing the composition of the first aspect of the present invention.

[0051] In one implementation, the method is carried out under room conditions of temperature and relative humidity.

[0052] The term "room temperature" refers to a temperature without heating or cooling, ranging from 15°C to 25°C.

[0053] The term "relative humidity room conditions" refers to the method being performed under the specified relative humidity of the air. In one embodiment, optionally combined with any of the embodiments provided above or below, the relative humidity is 50% to 80%. In one embodiment, optionally combined with any of the embodiments provided above or below, the relative humidity is 60% ± 5%.

[0054] In one embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the method comprising mixing an excess of a water-absorbing excipient by weight relative to the amount of live microorganisms expressed in volume units. When the composition of the invention contains more than one water-absorbing excipient, the expression "excess water-absorbing excipient by weight" means that the total amount of the water-absorbing excipient is excessive relative to the volume of the live microorganisms.

[0055] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the ratio between the amount of viable microorganisms, expressed in volume units, and the amount of water-absorbing excipients, expressed in weight units, includes a range from 0.1:1 to 0.99:1, preferably from 0.70:1 to 0.95:1. When the composition of the invention contains more than one water-absorbing excipient, the expression "amount of water-absorbing excipients" refers to the total amount of these excipients.

[0056] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the composition comprises a lubricant and the weight ratio between the absorbent and the lubricant comprises from 30:1 to 70:1, preferably from 40:1 to 60:1, and more preferably 50:1.

[0057] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the live microorganisms are fecal microbiota extracts.

[0058] Fecal microbiota extracts can be prepared by a method comprising the following steps: (a) providing fecal material obtained from a suitable donor; and (b) subjecting the fecal material to at least one processing step under certain conditions to produce a homogeneous composition of bacteria, archaea, fungi and viruses from the fecal material.

[0059] Fecal materials should be kept away from oxygen, for example, by covering the samples with an oxygen-depleting salt solution immediately after production, and by performing most of the treatment in an anaerobic environment (by using an anaerobic chamber or by flushing with, for example, Ar, N2 or CO2).

[0060] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below to homogenize, filter, and centrifuge the feces and brine. The supernatant is discarded, and the clumps are mixed with glycerol as a cryoprotectant to provide a fecal microbiota extract.

[0061] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the method comprising:

[0062] (a) Obtaining fecal microbiota extracts;

[0063] (b) Mix the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above.

[0064] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the method comprising:

[0065] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) centrifuging; and (a.4) mixing the clumps with a cryoprotectant;

[0066] (b) Mix the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above.

[0067] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the method comprising:

[0068] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) adding a cryoprotectant; and (a.4) centrifuging;

[0069] (b) Mix the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above.

[0070] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the method comprising:

[0071] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with brine and a cryoprotectant (e.g., glycerol), wherein the volume percentage of the cryoprotectant relative to the total volume of the solution is 5% to 15% or 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% (v / v); (a.2) filtering the solution; (a.3) adding a cryoprotectant, wherein the volume percentage of the cryoprotectant relative to the total volume of the solution is 10% to 50%, 10% to 40%, 15% to 35% or 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% (v / v); and (a.4) centrifuging; and (b) mixing the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above.

[0072] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the method comprising:

[0073] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing feces with brine and a cryoprotectant (e.g., glycerol), wherein the volume of the cryoprotectant is 10% (v / v) of the total volume of the solution; (a.2) filtering the solution; (a.3) adding a cryoprotectant at a volume of 20% (v / v) of the total volume of the solution; and (a.4) centrifuging; and (b) mixing the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above.

[0074] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, the method comprising:

[0075] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) centrifuging; and (a.4) mixing the clumps with glycerol;

[0076] (b) Mix the fecal microbiota extract with a water-absorbing excipient as defined in any of the embodiments provided above.

[0077] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0078] (a) Obtaining fecal microbiota extracts;

[0079] (b) Mix the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above;

[0080] (c) Adding one or more other pharmaceutically acceptable excipients; and

[0081] (d) The mixture obtained from encapsulation.

[0082] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0083] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) centrifuging; and (a.4) mixing the clumps with a cryoprotectant;

[0084] (b) Mix the fecal microbiota extract with a water-absorbing excipient as defined in any of the embodiments provided above;

[0085] (c) Adding one or more pharmaceutically acceptable excipients; and

[0086] (d) The mixture obtained from encapsulation.

[0087] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0088] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) centrifuging; and (a.4) mixing the clumps with a cryoprotectant;

[0089] (b) Mix the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above;

[0090] (c) Adding lubricant; and

[0091] (d) The mixture obtained from encapsulation.

[0092] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0093] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) centrifuging; and (a.4) mixing the clumps with a cryoprotectant;

[0094] (b) Mix the fecal microbiota extract with the cellulose-based excipients defined in any of the embodiments provided above;

[0095] (c) Adding one or more other pharmaceutically or veterinary excipients; and

[0096] (d) The mixture obtained from encapsulation.

[0097] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0098] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) centrifuging; and (a.4) mixing the clumps with a cryoprotectant;

[0099] (b) Mix the fecal microbiota extract with the cellulose derivatives defined above;

[0100] (c) Adding lubricant; and

[0101] (d) The mixture obtained from encapsulation.

[0102] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0103] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) centrifuging; and (a.4) mixing the clumps with a cryoprotectant;

[0104] (b) Mix the fecal microbiota extract with MCC;

[0105] (c) Adding stearate; and

[0106] (d) The mixture obtained from encapsulation.

[0107] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0108] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) centrifuging; and (a.4) mixing the clumps with a cryoprotectant;

[0109] (b) Mix the fecal microbiota extract with MCC, wherein an excess of the amount of MCC in weight is added relative to the amount of fecal microbiota extract in volume.

[0110] (c) Adding stearate; and

[0111] (d) The mixture obtained from encapsulation.

[0112] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0113] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline solution; (a.2) filtering the solution; (a.3) adding a cryoprotectant; (a.4) centrifuging; and (a.5) extraction;

[0114] (b) Mix the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above;

[0115] (c) Adding one or more other pharmaceutically acceptable excipients, such as lubricants; and

[0116] (d) The mixture obtained from encapsulation.

[0117] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0118] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with brine and a cryoprotectant (e.g., glycerol), wherein the volume percentage of the cryoprotectant relative to the total volume of the solution is 5% to 15% or 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% (v / v); (a.2) filtering the solution; (a.3) adding a cryoprotectant, wherein the volume percentage of the cryoprotectant relative to the total volume of the solution is 10% to 50%, 10% to 40%, 15% to 35% or 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% (v / v); (a.4) centrifugation; and (a.5) separating the extract;

[0119] (b) Mix the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above;

[0120] (c) Adding one or more other pharmaceutically acceptable excipients, such as lubricants; and

[0121] (d) The mixture obtained from encapsulation.

[0122] In another embodiment of the second aspect of the invention, this embodiment may optionally be combined with any of the embodiments provided above or below, wherein the method is performed to obtain a capsule, the method comprising:

[0123] (a) Obtaining a fecal microbiota extract by: (a.1) homogenizing the feces with saline and a cryoprotectant (e.g., glycerol), wherein the volume of the cryoprotectant is 10% (v / v) of the total volume of the solution; (a.2) filtering the solution; (a.3) adding a cryoprotectant at a volume of 20% (v / v) of the total volume of the solution; (a.4) centrifuging; and (a.5) separating the extract;

[0124] (b) Mix the fecal microbiota extract with one or more water-absorbing excipients as defined in any of the embodiments provided above;

[0125] (c) Adding one or more other pharmaceutically acceptable excipients, such as lubricants; and

[0126] (d) The mixture obtained from encapsulation.

[0127] By orally administering an effective amount of the composition containing microorganisms to a subject, the solid compositions of the present invention can be used for colonization of the gastrointestinal tract of any subject, such as a human recipient. Depending on the severity and current state of the disease, symptom, or illness, a recipient may be considered a patient, and the term "subject in need" includes both recipients and patients. Unless the context otherwise requires, all three terms are intended to refer to a human or animal that ingests one or more capsules of the present invention.

[0128] As used in this article, the term "object" refers to any mammal, including but not limited to livestock and other farm animals (such as cattle, goats, sheep, horses, pigs, and chickens), performance animals (such as racehorses), companion animals (such as cats and dogs), laboratory test animals, and humans. Typically, the object is a person.

[0129] Capsules containing this composition can treat, prevent, delay, or alleviate symptoms of diseases associated with dysbiosis (microbial imbalance or maladaptation in the body or mind). More specifically, the capsules of the present invention can be used to prevent or treat infections caused by Clostridium difficile, Salmonella spp., enteropathogenic E. coli, multidrug-resistant bacteria such as Klebsiella and E. coli, carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum beta-lactam-resistant Enterococci (ESBL), and vancomycin-resistant Enterococci (VRE).

[0130] In some embodiments, the subject suffers from inflammatory bowel disease (IBD), such as Crohn's disease, colitis (e.g., ulcerative colitis or microscopic colitis), or pouchitis; or irritable bowel syndrome or functional dyspepsia. In some embodiments, the subject suffers from liver disease, such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hepatic encephalopathy, primary sclerosing cholangitis (PSC), autoimmune hepatitis, or drug-induced liver injury. In some embodiments, the subject suffers from autoimmune diseases, such as celiac disease or eosinophilic esophagitis. In some embodiments, the subject suffers from hyperproliferative disorders of GI or malignancies, such as colorectal cancer / polyps, esophageal cancer, or Barrett's esophagitis. In some embodiments, the subject suffers from metabolic diseases such as metabolic syndrome, type 1 or type 2 diabetes, obesity, malnutrition or nutritional imbalance, or cardiovascular diseases (e.g., atherosclerosis). In other embodiments, the subject suffers from rheumatic diseases such as inflammatory arthritis (rheumatoid arthritis or RA, ankylosing spondylitis, psoriatic arthritis, IBD spondyloarthritis), fibromyalgia, chronic fatigue syndrome, or autoimmune and connective tissue diseases (e.g., systemic lupus erythematosus, scleroderma, and Sjogren's syndrome). In some embodiments, the subject suffers from vasculitis (e.g., polymyalgia rheumatica / giant cell arteritis or polyarteritis nodosa). In some embodiments, the subject suffers from mental disorders such as mood disorders (e.g., depression or bipolar disorder), anxiety disorders (e.g., generalized anxiety disorder, post-traumatic stress disorder), or developmental disorders (e.g., autism spectrum disorder, attention deficit hyperactivity disorder). In some embodiments, the subject suffers from one or more of colonic polyps, cysts, diverticulosis, constipation, intestinal obstruction, malabsorption syndrome, mucosal ulcers, and diarrhea. Other examples of diseases or conditions that can be treated with the capsules of the present invention are atopic dermatitis, rhinitis, and upper respiratory tract infection (URTI).

[0131] Because the microbiome of subjects suffering from chronic diseases or conditions often reverts to its inherently abnormal microbiome, repeated administration of the microbial community may be necessary to maintain clinical cure. Therefore, the compositions of the present invention containing microorganisms can be used as maintenance dose delivery. Maintenance dosing regimens can vary, including the microbial dose, frequency of administration, interval of administration, and duration of administration, and depend on the subject's disease and biological characteristics.

[0132] For example, treatment of chronic medical conditions may require a dose of about 5 to about 50 capsules for induction therapy, such as an administration of about 5 to about 40 capsules per dose. For example, the composition may be administered at a dose of about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 capsules per dose. The subject may be treated once or multiple times. For maintenance therapy, the capsules may be administered daily, or two to five times per week, or one to ten times per month. Maintenance therapy may last from weeks to months. For example, maintenance therapy may last from about two weeks to about six weeks (e.g., about one month), or it may last from about two months to about six months (e.g., about two months to four months) or even longer. A single “administration” refers to the capsules ingested within one day of treatment.

[0133] Throughout the specification and claims, the word "comprising" and variations thereof are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprising" covers situations where it means "consisting of...". Additional objects, advantages, and features of the invention will become apparent to those skilled in the art upon examination of the specification, or may be learned by practicing the invention. The following embodiments are provided by way of illustration and are not intended to limit the invention. Moreover, the invention covers all possible combinations of the specific and preferred embodiments described herein.

[0134] Example

[0135] Example 1

[0136] Transfer a 300g sample of fresh, refrigerated feces to a homogenizer bag containing 0.9% NaCl at a 1:10 ratio. Introduce it into a Stomacher 400 circulator (Seward Ltd., Sussex, UK) and homogenize at 230 rpm for 1 minute to obtain a slurry. Transfer the mixture to 50ml marked plastic tubes and add 10% pure glycerol (99%) before freezing at -80°C.

[0137] The process continued, with the sample thawed overnight at 4°C and 20% glycerol (99%) added. The mixture was then centrifuged at 400g for 20 minutes at 4°C (Heraeus Megafuge 16R Centrifuge, Thermo Fisher Scientific Inc., MA, USA) to remove sample debris. The supernatant was transferred to a high-resistance tube pre-filtered with a standard sieve to remove any possible debris, and this volume was centrifuged at 10000g for 30 minutes at 4°C (Sorvall Evolution RC Centrifuge, Thermo Fisher Scientific Inc., MA, USA) to obtain microbial flocculations. The supernatant was removed by decantation, and the flocculations were recovered with a scraper, avoiding any residual supernatant.

[0138] The pellet was divided into two portions for freeze-drying (for comparison purposes) and adsorption experiments (in this invention). Three identical equal portions were used for the experiment, each containing the equivalent of 50g of feces.

[0139] A. Preparation of capsules according to the present invention

[0140] A 9.3 mL aliquot was mixed with 10 g of microcrystalline cellulose (Vivapur®-101) to obtain the adsorbate. The formation of the adsorbate is understandable, as the appearance of the aliquot changed from a liquid to a "sawdust-like" texture. To aid encapsulation, 200 mg of magnesium stearate was further added to the adsorbate.

[0141] After obtaining the adsorbate, store the product overnight in a refrigerator at 4°C before packaging. Surround the product with a silica gel plate to reduce / eliminate humidity around the mixture in the refrigerator.

[0142] Finally, the adsorbent obtained was encapsulated into acid-resistant capsules No. 00 using a semi-automatic encapsulation machine, FagronLAB™ FG (Fagron Iberica, Barcelona, ​​Spain).

[0143] B. Preparation of capsules containing lyophilized microbiota (for comparative purposes)

[0144] Use Telstar LIOLAB 3 and follow the manufacturer's instructions for the freeze-drying process.

[0145] The resulting lyophilized material was then encapsulated into acid-resistant capsules (size 00) using a semi-automatic encapsulation machine, FagronLAB™ FG (Fagron Iberica, Barcelona, ​​Spain).

[0146] C. Bacterial viability analysis

[0147] Bacterial counts and viability from each replicate of original beaches, clumps, and capsules were measured using the LIVE / DEAD™ Baclight™ Bacterial Viability and Counting Kit (ThermoFisher Scientific, MA, USA) for flow cytometry and quantitative bacterial culture in Columbia agar (Becton Dickinson GmbH, Germany) containing 5% sheep blood, at 0, 1, and 3 months after sample storage at 4°C. The cytometer used was BDFACSCantoII (BD Biosciences, CA, USA), and the software was BD FACSDiva 8.0, according to the manufacturer's instructions. The Syto9:propidium iodide ratio was optimized to 1:1 using 0.1 µl of the sample to a final volume of 250 µl. An optimal dilution ratio of 1:10000 was found for the sample to be analyzed.

[0148] For flow cytometry analysis, the test aliquots of the capsule, along with the unencapsulated adsorbate and lyophilized material, were diluted 1:10000 with 0.9% NaCl solution as described above, and vigorously vortexed until a homogeneous liquid was obtained. Dilution in 0.9% NaCl yielded 10... -4 The bacterial dilution was 1:10000, with SYTO9 and propidium iodide in a 1:1 ratio (0.1 µl Syto9 and 0.1 µl propidium iodide, final volume 250 µL), and the kit contained 10 µl microspheres (1 / 2 dilution). Once results were obtained, the concentration of live bacteria was determined according to the equation in the protocol:

[0149]

[0150] D. Stability and morphological analysis

[0151] According to Section 2.5.12 of the Pharmacopoeia 9.4, the possible morphological changes and humidity of lyophilized capsules and capsules of the present invention, made from the mixture prepared as described above and kept at 4°C with or without silica gel, were determined using the Karl-Fischer method (Metrohm 899 coulometric meter). Hydranal-Coulomat AG was used as the reactant, and humidity was tested in 3 capsules for each condition. 100 mg of the contents of each capsule was taken as an aliquot and analyzed at a stirring rate of 10.

[0152] result

[0153] Three lyophilized capsules were obtained from each aliquot representing 50g of feces, while 14 to 17 capsules were obtained from the adsorbent capsules. No changes in capsule morphology (in terms of capsule length or width) or odor were observed during the 3-month study period.

[0154] Table 1. Results of bacterial culture and flow cytometry analysis of the capsules. The results show viability (i.e., the amount of viable microbiota).

[0155]

[0156] It can be seen that the capsules of the present invention are stable after three months at 4°C. When characterizing the water content, it was found that the capsules of the present invention contain a very high water content (see Table 2 below). Such a high content should negatively affect cell viability (in fact, those skilled in the art would expect cells to exhibit significant exponential growth).

[0157] In contrast, due to the inclusion of water-absorbing excipients, the bacterial population remained essentially unchanged from the start of the test.

[0158] The inventors repeated the same steps, but omitted the addition of magnesium stearate. They also concluded that the water-absorbing excipients provide the same "protective effect" for the microbiome.

[0159] Table 2. Humidity results of the capsules of the present invention

[0160]

[0161] Example 2

[0162] Materials / Methods

[0163] To analyze the concentration of live bacteria and microbial composition over a period of 6 months at 4°C, two samples (named M1 and M2) were obtained from 50 grams of feces from two different volunteers.

[0164] Each sample was processed according to the previously described protocol, and adsorbent capsules were obtained as follows:

[0165] (a) Vivapur-101 (i.e., microcrystalline cellulose) was added only as an adsorbent (samples M1V and M2V); and

[0166] (b) Vivapur-101 was added in combination with magnesium stearate as disclosed above (samples M1VS and M2VS).

[0167] The ratio between the volume of the aliquot sample (in "mL") and the amount of adsorbent and magnesium stearate (in "g") is essentially the same as that indicated in Example 1 above.

[0168] The bacterial concentration of the original sample was tested by flow cytometry, and the genome of the original sample was analyzed by 16S sequencing. After processing and centrifugation, adsorbates with two excipient combinations (M1V, M2V, M1VS and M2VS) were obtained.

[0169] For flow cytometry, the LIVE / DEAD BACLIGHT Staining and Counting Kit (ThermoFisher) was used. For genomic analysis, DNA was extracted using the PureLink™ Microbiome DNA Purification Kit (Invitrogen), and the V3 to V4 regions of the 16S rRNA gene were sequenced using the KAPA HiFiHotSart polymerase (Roche) on the Miseq platform (Illumina). Based on the obtained sequencing data, taxonomic composition was determined, and alpha diversity of the samples was also calculated to examine the product's stability in terms of microbial composition.

[0170] Alpha diversity refers to the species richness and diversity in each sample. For this determination, the Faith diversity index, or phylogenetic diversity (PD), calculated as the number of different species detected in a sample, is included in the phylogenetic distance between them in an evolutionary clade plot obtained using the qiime2 platform (www.qiime2). Diversity analysis helps to observe whether there is a loss of bacterial diversity during the production and storage of products.

[0171] Statistical analysis was performed using paired t-tests, with p < 0.05 considered statistically significant. Differences between results were tested using R version 3.6.2. Graphs were obtained using GraphPad Prism 8.02.

[0172] result

[0173] Bacterial concentration analysis

[0174] Table 3. Results of flow cytometry.

[0175]

[0176] The results showed that there was no significant difference between capsules at t=0 (p=0.125) and t=6 (p=0.029) in the MV group.

[0177] These results confirm that the adsorbent mixed with the fecal microbiota is the main reason for providing remarkable stability to bacterial activity for up to 6 months at 4°C.

[0178] Genome analysis

[0179] Once it is confirmed that the capsules of the present invention contain a large number of living cells and maintain this composition over time, the next step is to confirm whether the original bacterial diversity from the donor samples is also maintained. This is also relevant because the longer the bacterial diversity is maintained, the greater the efficacy.

[0180] Table 4. Alpha diversity results using the Faith index (PD).

[0181]

[0182] These results lead to the conclusion that when the capsules were formulated with the adsorbent (MV1), there was no significant difference between the original samples and the capsules at 6 months (p=0.922). This suggests that the inclusion of the adsorbent provides a suitable environment that avoids disruption and largely maintains the integrity and diversity of the original microbiome.

[0183] Table 4 also shows that the inclusion of additional excipients in the capsules according to the invention did not alter the behavior provided by the adsorbent, and no significant differences (M1VS and M2VS) were detected between the original sample and the capsules at 6 months. This indicates that the adsorbent provides a strong and beneficial stabilizing effect, as this stabilizing effect is not negatively affected even when other excipients are incorporated to optimize capsule manufacturing, and it essentially preserves the original diversity of the starting microbiome sample.

[0184] This application also includes, but is not limited to, the following implementation methods:

[0185] 1. A solid oral pharmaceutical composition comprising a pharmaceutically effective amount of live microorganisms and one or more pharmaceutically acceptable water-absorbing excipients, wherein the water content of the composition is from 0.5% to 30% of the total weight of the composition, as determined according to section 2.5.12 of the European Pharmacopoeia 9.4.

[0186] 2. The composition according to embodiment 1, wherein the composition is a single capsule.

[0187] 3. The composition according to any one of the preceding embodiments 1-2, wherein the live microorganisms are fecal microbiota.

[0188] 4. The composition according to any one of the preceding embodiments 1-3, wherein the water-absorbing excipient is selected from: cellulose-based excipients or pharmaceutically acceptable salts thereof; kaolinite; talc; palygorskite; sepiolite; and montmorillonite.

[0189] 5. The composition according to any one of the preceding embodiments 1-4, wherein the water-absorbing excipient is a cellulose ether derivative, a cellulose ester derivative, or a combination thereof.

[0190] 6. The composition according to any one of the foregoing embodiments 1-5, wherein the water-absorbing excipient is MCC.

[0191] 7. The composition according to any one of the preceding embodiments 1-6, wherein the water content accounts for 5% to 30% of the total weight of the composition.

[0192] 8. The composition according to any one of the preceding embodiments 1-7, wherein the composition comprises one or more other pharmaceutically acceptable excipients.

[0193] 9. The composition according to any one of embodiments 1-8, wherein the composition comprises:

[0194] - Cryoprotectant; or alternatively

[0195] - Lubricant; or alternatively

[0196] -Refrigeration protectants and lubricants.

[0197] 10. The composition according to any one of embodiments 1-9 above, comprising:

[0198] - Fecal microbiota, cellulose ether derivatives, stearates, and cryoprotectants; or alternatively,

[0199] - Fecal microbiota, cellulose ether derivatives, stearates, and glycerol; or alternatively,

[0200] - Fecal microbiota, cellulose ether derivatives, magnesium stearate, and glycerol; or alternatively,

[0201] - Fecal microbiota, MCC, stearate, and glycerol; or alternatively,

[0202] - Fecal microbiota, MCC, magnesium stearate and glycerol.

[0203] 11. A method for preparing an oral pharmaceutical composition according to any one of the foregoing embodiments, wherein the method comprises mixing live microorganisms with one or more water-absorbing excipients.

[0204] 12. The method according to embodiment 11, wherein the method includes mixing an excess of water-absorbing excipient by weight relative to the amount of live microorganisms expressed in volume units.

[0205] 13. The method according to any one of embodiments 11-12 further includes adding a lubricant, wherein the weight ratio of water absorbent to lubricant is 30:1 to 70:1.

[0206] 14. A solid oral pharmaceutical composition according to any one of embodiments 1-10 for therapeutic purposes.

[0207] 15. Use of water-absorbing excipients for stabilizing live microorganisms in pharmaceutical compositions.

[0208] Citation List

[0209] Patent documents

[0210] KR20080059605.

[0211] Non-patent literature

[0212] Section 2.5.12: Water: Semi-micro determination, “European Pharmacopoeia 9.4”, 2018, p. 5107

Claims

1. A solid oral pharmaceutical composition comprising a pharmaceutically effective amount of live microorganisms and one or more pharmaceutically acceptable water-absorbing excipients, wherein the water content of the composition is from 0.5% to 30% of the total weight of the composition, as determined according to section 2.5.12 of the European Pharmacopoeia 9.

4.

2. The composition according to claim 1, wherein, The composition is a single capsule.

3. The composition according to any one of claims 1-2, wherein, The live microorganisms mentioned are fecal microbiota.

4. The composition according to any one of claims 1-3, wherein, The water-absorbing excipient is selected from: cellulose-based excipients or their pharmaceutically acceptable salts; kaolinite; talc; palygorskite; sepiolite; and montmorillonite.

5. The composition according to any one of claims 1-4, wherein, The water-absorbing excipient is a cellulose ether derivative, a cellulose ester derivative, or a combination thereof.

6. The composition according to any one of claims 1-5, wherein, The water-absorbing excipient is MCC.

7. The composition according to any one of claims 1-6, wherein, The water content is 5% to 30% of the total weight of the composition.

8. The composition according to any one of claims 1-7, wherein, The composition comprises one or more other pharmaceutically acceptable excipients.

9. The composition according to any one of claims 1-8, wherein, The composition comprises: - Cryoprotectant; or alternatively - Lubricant; or alternatively -Refrigeration protectants and lubricants.

10. The composition according to any one of claims 1-9, comprising: - Fecal microbiota, cellulose ether derivatives, stearates, and cryoprotectants; or alternatively, - Fecal microbiota, cellulose ether derivatives, stearates, and glycerol; or alternatively, - Fecal microbiota, cellulose ether derivatives, magnesium stearate, and glycerol; or alternatively, - Fecal microbiota, MCC, stearate, and glycerol; or alternatively, - Fecal microbiota, MCC, magnesium stearate and glycerol.

11. A method for preparing an oral pharmaceutical composition according to any one of the preceding claims, wherein, The method includes mixing live microorganisms with one or more water-absorbing excipients.

12. The method according to claim 11, wherein, The method includes mixing an excess of water-absorbing excipient by weight relative to the amount of live microorganisms expressed in volume units.

13. The method according to any one of claims 11-12, further comprising adding a lubricant, wherein the weight ratio of water absorbent to lubricant is 30:1 to 70:

1.

14. A solid oral pharmaceutical composition according to any one of claims 1-10 for therapeutic use.

15. Use of water-absorbing excipients for stabilizing live microorganisms in pharmaceutical compositions.

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