The present invention relates to the manufacture of a pellet formulation comprising an adsorbent and one or more excipients providing binder and disintegrating properties.

Applications WO2006122835, W02007132022 and WO2011104275 describe formulations for the controlled delivery of an adsorbent into the lower intestine of mammals. These formulations comprise an adsorbent-containing core and an external coating designed to release the adsorbent contained into the core to the lower gastro-intestinal tract. The present applicant has developed such a formulation, referred to as DAV 132, as a novel microbiota protective therapy to prevent antibiotic-induced dysbiosis. Co-administered with antibiotics, it has demonstrated its ability to selectively and safely capture antibiotics. Multiple clinical trials have already been performed successfully in healthy volunteers and patients.

In WO2011104275, manufacture of the adsorbent core of this kind of formulations was done by wet granulation followed by extrusion spheronization. This previously described method provides high quality formulations with pellet size in the 0.5 - 1.0 mm range. However, specific uses of the formulations, such as their administration via tube feeding and pediatric uses would advantageously benefit from the production of smaller pellets. In addition, the manufacture process should allow scaling- up of the production for industrial purposes.

The present invention relates to the development of a new process meeting these needs.

More specifically, the present invention relates to the manufacture of a dosage form of an adsorbent, said formulation comprising an adsorbent-containing core and an external coating designed to release the core at a desired part of the intestine.

Accordingly, it is herein disclosed a method for the manufacture of adsorbent-containing pellets, wherein the pellets comprise: a) an adsorbent; and b) one or more excipients providing binding and disintegration properties; and wherein said manufacture is carried out in a fluid bed apparatus.

The term “adsorbent” designates any compound or material that can adsorb a substrate, typically by physico-chemical binding between the adsorbent surface and the substrate(s) to be adsorbed. Adsorbents may be specific or non-specific. Preferred adsorbents for use in the invention are pharmaceutical grade adsorbents, best suited for use in humans or animals for pharmaceutical or veterinary applications.

Examples of adsorbents suitable for use in the present invention include, without limitation, activated charcoal (also referred to as activated carbon, active carbon, or active charcoal); clays, including bentonite, kaolin, montmorrillonite, attapulgite, halloysite, laponite, and the like; silica, including colloidal silica (Ludox® AS-40 for example), mesoporous silica (MCM41), fumed silica, zeolites and the like; talc; cholesteramine and the like; polystyrene sulfonates and the like; mono and polysulfonated resins; as well as other resins such as those used for bacteriologic testing such as BACTEC® resins.

Preferred adsorbents are activated charcoals which are of pharmaceutical grade. In a particular embodiment, the adsorbent is activated charcoal, more particularly an activated charcoal having a specific surface area above 600 m ² /g, in particular above 800 m ² /g, in particular above 1000 m ² /g, in particular above 1200 m ² /g, in particular above 1400 m ² /g, in particular above 1600 m ² /g, even more particularly above 1800 m ² /g. The activated charcoal may be of vegetal, mineral or synthetic origin, its surface being optionally modified by a physical or chemical treatment. In a particular embodiment, the activated charcoal is of vegetal origin. In a particular embodiment, the activated charcoal is derived from wood. In a particular embodiment, the activated charcoal is derived from peat. In a particular embodiment, the activated charcoal is derived from coconut husks. In a particular embodiment, the activated charcoal is derived from different sources mixed together such as peat and coconut husks. In a particular embodiment, the activated charcoal is characterized by a European molasses number (of note the European molasses number is inversely related to the North American molasses number) which is preferably higher than 100, even more particularly greater than 200, even more particularly greater than 300, even more particularly greater than 400, even more particularly greater than 500, even more particularly greater than 600. In a particular embodiment, the activated charcoal has a phenazone number (measured according to the EU Pharmacopeia) greater than 10 g/100 g, even more particularly greater than 20 g/100 g, even more particularly greater than 30 g/100 g, even more particularly greater than 40 g/100 g, even more particularly greater than 50 g/100 g, even more particularly greater than 60 g/100 g. In a particular embodiment, the activated charcoal is characterized by a density between 0.05 and 0.8, even more particularly between 0.1 and 0.6, even more particularly between 0.15 and 0.5, even more particularly between 0.2 and 0.4.

The amount of the adsorbent, in particular of activated charcoal, will range from about 1 % to about 99 % by weight of the total pellet to be produced, preferably from about 50 % to about 95 %, most preferably from about 65 % to about 95%, in particular from about 80 % to about 95 % by weight of the pellet to be produced. In a further particular embodiment, the amount of the adsorbent is of about 85 % by weight of the total pellet to be produced. As used herein with respect to a numerical value, the term "about" means ±10 % of said numerical value, in particular ±5 % of said numerical value, more particularly ±2 % of said numerical value, even more particularly ±1 % of said numerical value.

The pellets manufactured thanks to the invention contain at least one excipient providing binding and disintegration properties. These properties can be provided by one excipient having both properties, by several excipients each having both properties, or by several excipients each having one or both of the properties (such as by two excipients, one having binding properties, and the other having disintegration properties).

In a preferred embodiment, the pellet to be produced comprises an excipient having both binding and disintegration properties. Of course, inclusion of such an excipient does not exclude the possibility of including into the pellet further excipients, such as anti-adherents, binders, fdlers, diluents, flavours, coloration agents, lubricants, glidants, preservatives, sorbents and/or sweeteners.

In a preferred embodiment, the excipient having both binding and disintegration properties is a carrageenan. In a particular embodiment, the carrageenan can be selected from kappa-carrageenan, iota- carrageenan and lambda-carrageenan, and mixtures thereof. In a particular embodiment, the carrageenan is kappa-carrageenan.

In a particular embodiment, the pellet contains a mixture of excipient, such as a mixture of an excipient having both binding and disintegration properties as described above, and polyvinyl alcohol. In a particular embodiment, the polyvinyl alcohol as a molecular weight comprised between 5000 and 250000, such as between 9000 and 205000. In yet another particular embodiment, the pellet contains a mixture of a carrageenan and polyvinyl alcohol. In another embodiment, the pellet contains a mixture of a kappa carrageenan and polyvinyl alcohol.

The amount of the excipient(s) will depend both on the amount of adsorbent to be formulated, and on the binding and disintegrating properties sought. In a particular embodiment wherein the one or more excipients is carrageenan, in particular kappa-carrageenan, the amount of carrageenan will range from about 1 % to about 99 % by weight of the total pellet to be produced, preferably from about 5 % to about 50 %, most particularly from about 5 % to about 35%, in particular from about 5 % to about 25 %, more particularly about 10 to about 20% by weight of the pellet to be produced. In a further particular embodiment, the amount of carrageenan is of about 15 % by weight of the total pellet to be produced. In a particular embodiment wherein the one or more excipients is carrageenan (in particular kappa carrageenan) and polyvinyl alcohol, said excipients may be present at a weight ratio of 99: 1 to 1:99, in particular 99: 1 to 25:75, such as a ratio of 75:25 to 25:75, more particularly of 50:50. In a further particular variant of this embodiment, the amount of the mixture of carrageenan and polyvinyl alcohol in the pellet ranges from about 1% to about 99% by weight of the total pellet to be produced, preferably from about 5 % to about 50 %, most particularly from about 5 % to about 35%, in particular from about 5 % to about 25 %, more particularly about 10 to about 20% by weight of the pellet to be produced, such as in an amount of 15% by weight of the pellet to be produced.

As used herein with respect to a numerical value, the term "about" means ±10 % of said numerical value, in particular ±5 % of said numerical value, more particularly ±2 % of said numerical value, even more particularly ±1 % of said numerical value.

In one particular embodiment, the adsorbent is activated charcoal, and the one or more excipient providing binding and disintegration properties is kappa-carrageenan. Preferably, the amount of kappa- carrageenan is between about 2.5% and about 25%, more preferably between about 5% and about 20%, in particular between about 7.5 and about 15% by weight of the pellet to be produced. According to a specific embodiment of the invention, the amount of kappa-carrageenan is about 15% by weight of the pellet to be produced. Preferably, the amount of activated charcoal is between about 75% and about 95%, more preferably between about 80% and about 90%, by weight of the pellet to be produced. According to a specific embodiment of the invention, the amount of activated charcoal is about 85% by weight of the pellet to be produced. For example, the pellet to be produced may contain between about 5% and about 25% of kappa-carrageenan and between about 75% and about 95% of activated charcoal, by weight of the pellet to be produced. In another example, the pellet to be produced may contain between about 10% and about 20% of kappa-carrageenan and between about 80% and about 90% of activated charcoal, by weight of the pellet to be produced. In a specific embodiment, the pellet to be produced may contain about 85% of activated charcoal and 15% of kappa-carrageenan, by weight of the pellet to be produced.

In another particular embodiment, the adsorbent is activated charcoal, the one or more excipient providing binding and disintegration properties is kappa-carrageenan and the pellet further comprises polyvinyl alcohol. Preferably, the amount of the mixture of kappa-carrageenan and polyvinyl alcohol is between about 3% and about 25%, more preferably between about 4% and about 20%, more preferably between about 10% and about 20%, in particular between about 6% and about 8% by weight of the pellet to be produced. According to a specific embodiment of the invention, the amount of the mixture of kappa-carrageenan and polyvinyl alcohol is about 15% by weight of the pellet to be produced. Preferably, in this embodiment wherein the one or more excipient is a mixture of kappa-carrageenan and polyvinyl alcohol, the amount of activated charcoal is between about 75% and about 97%, more preferably between about 85% and about 95%, more preferably between about 90% and 94% by weight of the pellet to be produced. According to a specific embodiment of the invention, wherein the one or more excipient is a mixture of kappa-carrageenan and polyvinyl alcohol, the amount of activated charcoal is about 92.5 % by weight of the pellet to be produced.

The adsorbent-containing formulation of the invention is manufactured by fluid pelletization in a fluid bed apparatus.

Several methods of fluid bed pelletization can be implemented, such as Wurster and rotor technologies. In a particular embodiment, a jet bed or a spouted bed is used. In another particular embodiment, the fluid bed pelletization process is a batch-wise or continuous process. Preferably, the invention involves the use of a continuous fluid bed pelletization process, most preferably a continuous spouted bed pelletization process. Illustrative equipments include, without limitation, the ProCell Technology marketed by Glatt.

The starting material may be a suspension (otherwise herein referred to as "solid suspension") comprising the adsorbent and the one or more excipient(s), in a pelletizing liquid. The suspension may further comprise any other ingredient as defined above. The pelletizing liquid may be selected among a number of suitable liquids, such as water, organic solvents, and mixture thereof, such as a water/alcohol mixture. Accordingly, in a particular embodiment, the starting material is an aqueous suspension comprising the adsorbent and the one or more excipient(s). The suspension may be prepared by mixing to homogeneity powders of the adsorbent and of the one or more excipient(s) in the above described relative amounts, with the desired amount of solvent (e.g. water) to reach a defined concentration of the solid (solid concentration). In a particular embodiment, the solid concentration in the suspension is comprised from about 3% to about 15% w/v, in particular from about 4% to about 12%, more particularly from about 5% to about 10%, such as from about 6% to about 9% w/v. In a particular embodiment, the solid concentration in the solid suspension is of about 6%, about 7%, about 8% or about 9% w/v. In a further particular embodiment, the solid concentration in the solid suspension is at a concentration of about 6% or about 8% w/v. Optionally, the solid suspension may be sieved through a sieve to remove potential aggregates, such as a 100 pm - 1000 pm sieve, for example a 100 pm sieve, a 200 pm sieve, a 300 pm sieve, a 400 pm sieve, a 500 pm sieve, a 600 pm sieve, a 700 pm sieve, a 800 pm sieve, a 900 pm sieve or a 1000 pm sieve. Preferably, the solid suspension is kept under constant stirring from initial mixing of its components until its use in the fluid bed equipment.

In a particular embodiment, dry raw material powder (i.e. a mixture of dry powders of the adsorbent and of the one or more excipient(s), in the above described proportions) may first be directly introduced into the equipment, as an initiator of pellet growth, before introduction of the solid suspension. Alternatively, introduction of the solid suspension into the equipment is done without initiation of pellet growth. The solid suspension is introduced into the fluid bed pelletizing equipment according to procedures known in the art. For example, the solid suspension may be pumped and sprayed to the fluid bed equipment through a peristaltic pump. Depending on the selected equipment, the solid suspension may be sprayed from the bottom or the top of the fluid bed chamber. In a particular embodiment implementing spouted bed, the solid suspension is sprayed from the bottom of the fluid bed chamber.

Fluid bed process parameters may vary, but illustrative parameters are provided below with respect to spouted bed technology:

- air flow: from 50 to 300 m ³ /h, such as from 75 to 200 m ³ /h;

- inlet air temperature: from 50°C to 300 °C, such as from 75°C to 250°C;

- spray pressure: from 0.3 to 10 bar, such as from 0.5 to 7.5 bar;

- spray rate: from 5 to 200 g/min, such as from 10 to 150 g/min.

According to the method described herein, adsorbent-containing pellets of controlled size are formed. Advantageously, pellets having a mean size comprised between 0.1 and 0.9 mm, in particular between 0.2 and 0.8 mm, more particularly between 0.3 and 0.7 mm can be obtained thanks to the present method, which is significantly lower than the pellet size obtained with previous extrusion spheronization processes.

Solvent (such as water) remaining in the pellets may be removed during an optional drying step. Such drying step may be conducted in the same or a different equipment, preferably the same. Drying parameters may vary to a large extent and may comprise both a controlled inlet temperature (for example between 50 and 150°C) and a controlled chamber temperature (for example between 35 and 75 °C). Drying may be carried out until a targeted moisture content is reached, such as a moisture content below 10%, in particular below 9%, 8%, 7%, 6%, 5%, or even below 4%.

Furthermore, and independently of a target moisture content, drying may be carried out to reach suitable disintegration properties. Therefore, in a particular variant of this embodiment, drying may be carried out during up to 3 hours.

In another optional step, the pellets may be further polished to reduce or minimize the number of tips, peaks and edges at the surface of the pellets. Such polishing step may be carried out by causing the produced pellets to collide with each other. This step can be conducted in a continuous process into the fluid bed equipment used in the previous step(s), or in any other suitable equipment known to those skilled in the art.

In a particular embodiment, the adsorbent-containing pellets are then further coated with an external coating suitable to release the pellet and its adsorbent content in a desired part of the gastro-intestinal tract, such as the lower gastro-intestinal tract (i.e. in the late ileum, the caecum and/or the colon). Such pellet coated with an external coating may be referred to as a coated pellet in the present application.

In a particular embodiment, pellets formed at the previous step being first collected and then introduced in a suitable equipment, for further coating.

Examples of suitable coatings include pH-dependent enterosoluble polymers, azopolymers, disulphide polymers, and polysaccharides, in particular amylose, pectin (e.g. pectin crosslinked with divalent cations such as calcium pectinate or zinc pectinate), chondroitin sulphate and guar gum. Representative pH-dependent enterosoluble polymers include cellulose acetate trimellitate (CAT), cellulose acetate phthalate (CAP), acrylic polymers, methacrylic polymers, anionic copolymers based on methylacrylate, methylmethacrylate and methacrylic acid, hydroxypropyl methylcellulose phthalate (HPMCP), hydroxypropylmethylcellulose acetate succinate (HPMCAS), methacrylic acid and ethyl acrylate copolymers, methacrylic acid and methyl methacrylate copolymers in a 1 : 1 molar ratio, methacrylic acid and methyl methacrylate copolymers in a 1:2 molar ratio, polyvinyl acetate phthalate (PVAP) and shellac resins. Particularly preferred polymers include shellac, anionic copolymers based on methyl acrylate, methyl methacrylate and methacrylic acid, such as poly(methyl acrylate-co-methyl methacrylate-co-methacrylic acid) in a 7:3: 1 molar ratio, as well as methacrylic acid and methyl methacrylate copolymers in a 1:2 molar ratio. Ideally, the polymer dissolves at a pH equal to 6.0 and above, preferably 6.5 and above. Suitable coatings may also be obtained by mixing the polymers and copolymers aforementioned. In another embodiment, suitable coatings are time-dependent coatings or based on time-dependent polymers such as mixture of ethylcellulose polymers with alginate sodiums.

In a particular embodiment, the external coating is an anionic copolymer based on methyl acrylate, methyl methacrylate and methacrylic acid, such as poly(methyl acrylate-co-methyl methacrylate-co- methacrylic acid) 7:3: 1, e.g. Eudragit® FS30D.

As mentioned above, the adsorbent-containing pellets manufactured thanks to the invention have a mean particle size comprised between 0.1 and 0.9 mm, in particular between 0.2 and 0.8 mm, more particularly between 0.3 and 0.7 mm. Such pellets were never reported before. They can be advantageously be used in a number of applications. For example, thanks to the smaller size of these pellets, tube feeding and pediatric achieved more efficiently. Accordingly, a further aspect of the present invention relates to a composition comprising an adsorbent and one or more excipient(s), wherein the composition is in the form of a pellet having a mean particle size comprised between 0.3 and 0.7 mm. In particular embodiments, the adsorbent and the one or more excipient(s) are present in said composition in an amount as described in any of the embodiments described above. Preferably, the adsorbent is activated charcoal and the one or more excipient(s) is carrageenan, more particularly kappa-carrageenan. In another preferable embodiment, the composition comprises about 85% of activated charcoal and about 15% of kappa-carrageenan. In another embodiment, the composition is coated with an external coating as described above. Preferably, the external coating is an anionic copolymer based on methyl acrylate, methyl methacrylate and methacrylic acid, such as poly(methyl acrylate-co-methyl methacrylate-co- methacrylic acid) 7:3: 1, e.g. Eudragit® FS30D.

Once the coated pellets have been produced, they may be formulated into individual dosage units for administration of an adsorbent for therapeutic and/or prophylactic purposes such as capsules, sachets, stick-packs and tablets. They can alternatively be introduced into containers (e.g. glass or plastic bottles) comprising an amount of pellets sufficient for the user to take multiple doses for the intended use, such as by using a dosing spoon or dosing pipet.

The coated pellet of the invention may be used in a number of applications described in W02006122835, W02007132022, WO2011104275, W02017060178, EP18187408 and EP18187409. More generally, the coated pellet of the invention can be used in a method to protect the intestinal microbiota from antibiotic -induced disruption. Therefore, the invention also relates to a coated pellet as herein described, for use in a method to:

- eliminate or reduce the side effects in the intestine, in particular in the colon, of pharmaceutical agents that are administered as a treatment for a disorder, but that have side effects when they, or a metabolite or derivative thereof, reach the late ileum, the caecum or the colon;

- to eliminate or reduce the antibiotic -associated adverse effects of antibiotic agents, in particular for eliminating or reducing the emergence of antibiotic resistance or for eliminating or reducing diarrhea;

- suppress or reduce the pathogenicity or virulence of a bacterium of the genus Clostridium or Clostridioides,'

- suppress or delay the production of toxins by a bacterium of the genus Clostridium or Clostridioides, and/or to suppress or delay the germination of spores of a bacterium of the genus Clostridium or Clostridioides, and/or to suppress or delay the sporulation of a bacterium of the genus Clostridium or Clostridioides,'

- to treat or prevent a cancer;

- to improve the efficacy of an anticancer agent such as an immuno-oncology agent;

- to treat, prevent, delay or reduce the risk or severity of Graft-Versus-Host disease; and

- to reduce post-transplant mortality in subjects receiving hematopoietic stem cell transplant.

More generally, the coated pellet of the invention can be used in a method to protect the intestinal microbiota from antibiotic-induced disruption. In a particular embodiment, the coated pellets of the invention are used in a method to prevent Clostridium or Clostridioides difficile infections or emergence of antibiotic-resistant bacteria. In another embodiment, the coated pellets of the invention can be used to improve the efficacy of immunotherapies for patients with cancer.

The invention will now be illustrated with reference to the following non-limiting examples.

EXAMPLES

Example 1: manufacturing of pellets of adsorbent with a spouted bed

Material and methods

The pellet cores contained at least 85% of activated charcoal (AC) and 15% of binder/disintegrating comprising k-carrageenan and the polyvinyl alcohol (PVA). These binders were used individually or mixed in ratio from 100:0 (k-carrageenan exclusively) to 50:50 w/w. The following range of formulation were produced.

AC and the excipients used as binder are mixed in purified water to form a solid dispersion before starting the pelletization. The main part of the water is removed during the pelletization process, and thus this amount is not considered in the formulation as a raw material.

The equipment used for the pelletization of FAC pellets is a continuous fluid bed system. Powder is generated by spray drying; the powder is stepwise agglomerated to create pellets. The growth continues until spheroid pellets of the desired size are obtained.

The pelletization process is composed of 3 main steps:

• Preparation of the suspension: An aqueous suspension of AC and binder in the ratio of the formula are mixed with the desired amount of water to reach a pre-defined solid concentration and stirred until a homogeneous dispersion is obtained.

• Pelletization: The suspension is pumped to the fluid bed system through a peristaltic pump, sprayed from the bottom of the chamber and dried to form ever-growing aggregates. This step allows the formation of spheroid pellets of the same size range. Pellets are maintained in the chamber of the fluid bed process until the targeted particle size distribution (PSD) is reached. The zig-zag sifter allows the discharge of pellets of the appropriate size.

• Drying: After the pelletization, the pellets are dried in a fluid bed dryer.

Main process parameters of all trials were in the ranges described in the table hereafter:

The pellets obtained with the process described above were compared to pellets previously obtained by wet granulation followed by extrusion spheronization in order to ensure the pellets obtained are appropriate.

The morphological properties of pellets are examined with 2 measures: Particle Size Distribution (PSD) and aspect ratio. The PSD is measured through image analysis is expressed in mm or pm as the size distribution for the 1 ^st decile of the distribution (DIO), the median of the distribution (D50) and the 9 ^th decile of the distribution (D90). For example, a value D50 = 100 pm means that half of the pellets have a diameter above 100 pm and half below that value. The shape of the pellets was also characterized by its aspect ratio: it was measured through a visual examination of photos of the pellets and measurements of the length and width of the pellets and the ratio of the length and width being computed.

Each formulation was also tested for its adsorption capacity of a model antibiotic (ABX). The adsorption capacity reflects the product performance, considering that the in vivo action of the product is the adsorption of the residual antibiotic in the intestine. In this in-vitro test, the adsorption capacity is the result of the disappearance of the ABX in a medium in defined conditions. At the end of the test, samples are analyzed in high-performance liquid chromatography in order to measure the remaining ABX concentration in the media. The result is expressed as the quantity of ABX (in mg) adsorbed per quantity of AC in the medium (in g).

To be compliant, the produced pellets should respect the following specifications:

All the trials presented in the table hereafter led to obtain pellets at the end of the manufacturing process as described above.

From all these batches, adsorption capacity analyses were performed. Results are presented in the table below.

All batches obtained during the development of the process were compliant with the specifications. The adsorption properties of AC pellets obtained with the new process were equivalent and even slightly better than the pellets obtained by extrusion.

In terms of PSD, the current performances obtained by extrusion-spheronization were around 0.4 to 0.8 mm. Results obtained by fluid bed pelletization were thus compared to this target. The PSD values of all dry samples of the formulation study are presented in the table hereafter.

Results showed that various profile of PSD were obtained with the different batches. Data from batches Mini-B/003and Mini-B/011, produced with the same formula, led to different PSD due to adjustments of process parameters (inlet air temperature, inlet air volume, suspension concentration in solids). This proved that a specific target of PSD can be achieved by adaptation of the process conditions.

Batch Mini-B/011, formulated with 92.5% of AC of carrageenan was considered as the batch leading to the performances as close as possible to the previous prototype obtained in extrusion spheronization. These results showed that the fluid bed pelletization process described can be used to achieve high concentrated AC pellets with similar and even better properties than those obtained with a previously developed extrusion spheronization process. Indeed, surprisingly this process allowed to increase the AC content from 85% to 92.5% in the formula without impacts on main quality attribute of the AC spheroid pellets.