Method for controlling administration of an active substance to the gastrointestinal tract - Patents.com

Abstract

The present disclosure is directed to methods of providing effective oral administration of an active agent, including a pharmaceutical ingredient, a dietary supplement, an enzyme, or a probiotic, using a delivery system for optimal bioactivity and absorption of the active agent by the mammal to which the active agent is delivered.

Description

[Technical field]

[0001] Related Applications This application is based on and claims priority to U.S. Provisional Patent Application No. 63 / 214,438, filed June 24, 2021, which is incorporated herein by reference. [Background technology]

[0002] Oral administration of active substances such as pharmaceutical ingredients, dietary supplement ingredients, or probiotics is generally the preferred method for administering active substances to mammals due to its convenience, potential controlled release, and user compliance.Despite these advantages, many challenges are associated with oral administration of active substances, such as product performance, sufficient and efficient dosing of active substances, and viability of active substances in the gastrointestinal tract activity.

[0003] In the upper gastrointestinal tract (GIT), orally administered pharmaceutical ingredients, nutraceutical ingredients, and probiotics are susceptible to degradation due to the harsh acidic conditions in the stomach and gastric enzymes (i.e., pepsin). In the duodenum, pancreatic enzymes (i.e., lipase, trypsin, amylase, peptidase) and bile salts can significantly affect the stability of these ingredients, especially the viability of probiotics. Different transit times, pH profiles, and enzyme levels have been described during fasted or fed conditions, necessitating adjustment of the dosage form of oral entities for better efficacy and performance.

[0004] Therefore, immediate release formulations should be avoided when pH-sensitive active substances are delivered orally. For example, probiotics, which are live microorganisms, only confer a health benefit to the host when administered at appropriate levels and may perform less well if strain viability is reduced during GIT transit, for example, due to low pH. Nutritional supplements such as flavonoids, carotenoids, hydroxycinnamoyl acid, or vitamin C can also be highly degraded (80-91%) during gastrointestinal digestion, while bioactive substances such as proteins and peptides can be damaged by the action of pepsin and trypsin degradation, thus significantly reducing their activity.

[0005] Different strategies, including tablet coating or bioactive encapsulation, have been developed to provide suitable formulations for acid-sensitive products. Tablets have the disadvantages of low compressible content, slow dissolution, or bitter taste. In addition, during the early stages of drug development, limited amounts of drug availability may hinder the development of coated pellet or tablet formulations. Therefore, certain capsule polymers, such as cellulose derivatives or acrylic / methacrylic acid derivatives, may provide better solid dosage forms and also offer the possibility of targeting the delivery of liquid or semi-solid formulations to the small or large intestine. Thus, capsule technology has made great progress in the past few years, providing an economically convenient alternative for pharmaceutical, nutraceutical, and probiotic formulations, as well as targeted release of entities. Summary of the Invention

[0006] In general, the present disclosure is directed to methods of providing effective oral administration of an active agent, including a pharmaceutical ingredient, a dietary supplement, an enzyme, or a probiotic, using a delivery system for optimal bioactivity and absorption of the active agent by the mammal to which the active agent is delivered.

[0007] In a first embodiment, the disclosure is directed to a method for providing an effective oral administration of an active agent to a mammal, the active agent being delivered to the digestive tract of the mammal. The method includes preparing a delivery system, the delivery system including an outer capsule having an outer shell wall and an inner chamber, and an inner capsule having an outer shell wall and an inner compartment. The inner capsule is located within the inner chamber of the outer capsule, the inner capsule being acid-resistant and containing an active agent. The active agent is present within the inner compartment of the inner capsule. The delivery system is orally administered to the mammal, the delivery system delivering the active agent in an effective amount to the intestine of the mammal.

[0008] In a second embodiment, the disclosure is directed to a method of modifying gut microbiome and colonization by administering an active ingredient to the gut. The method includes preparing a delivery system. The delivery system includes an outer capsule having an outer shell wall and an inner chamber, and an inner capsule having an outer shell wall and an inner compartment. The inner capsule is located within the inner chamber of the outer capsule, and the inner capsule may be formulated to be acid-resistant. A probiotic active ingredient is present within the inner compartment of the inner capsule. The delivery system is orally administered to a mammal, and the delivery system delivers the probiotic active in an effective amount to the gut of the mammal. The active ingredient improves the microbiome or colonization of healthy bacteria in the gut.

[0009] In a further aspect of the disclosure, in the delivery system, the outer capsule comprises an HPMC hard capsule.

[0010] In a further aspect of the present disclosure, in the delivery system, the inner capsule comprises an acid-resistant HPMC hard capsule.

[0011] In another embodiment of the present disclosure, the inner capsule comprises a capsule comprising HPMC and gellan gum. In one particular embodiment, the gellan gum is present in an amount of about 4 parts to 15 parts per 100 parts HPMC.

[0012] In another embodiment of the disclosure, the HPMC outer capsule comprises thermogelling HPMC.

[0013] In a further embodiment of the present disclosure, the outer capsule is an acid-resistant capsule. In one aspect, the outer acid-resistant capsule is an acid-resistant HPMC hard capsule. In a particular embodiment, the outer capsule is a capsule comprising HPMC and gellan gum. The gellan gum is present in an amount of about 4 parts to 15 parts per 100 parts of HPMC.

[0014] In one embodiment, the active agent comprises a probiotic.

[0015] In a different embodiment of the present disclosure, the inner capsule and the outer capsule each comprise an acid resistant capsule, each comprising HPMC and gellan gum.

[0016] In another embodiment, the method provides for delivery of an active agent to the colon in an amount at least 10 times greater than a capsule that dissolves in the stomach or small intestine. In a further embodiment, the active agent is delivered to the colon in an amount at least 20 times greater than a capsule that dissolves in the stomach or small intestine, and even at least 30 times greater than a capsule that dissolves in the stomach or small intestine.

[0017] Other features and aspects of the disclosure are discussed in more detail below. [Brief description of the drawings]

[0018] [Figure 1] 1 shows a delivery system that can be used in the methods of the present disclosure having a capsule-in-capsule configuration. [Diagram 2] 1 shows a delivery system that can be used in the methods of the present disclosure having a triple capsule configuration. [Diagram 3] 1 illustrates a delivery system that can be used in the methods of the present disclosure having a multi-capsule configuration. [Figure 4A] 1 shows the pH profile used in the examples under fed conditions. [Figure 4B]1 shows the pH profile used in the examples under fasting conditions. [Diagram 5] Figure 5A shows the effect of capsule configuration on caffeine release in the stomach and small intestine under simulated fasting conditions. Figure 5B shows the effect of capsule configuration on caffeine release in the stomach and small intestine under simulated fasting conditions. Figure 5C shows the effect of capsule configuration on caffeine release in the stomach and small intestine under simulated fasting conditions. Figure 5D shows the effect of capsule configuration on caffeine release in the stomach and small intestine under simulated fasting conditions. [Figure 6] Figure 6A shows the effect of capsule configuration on caffeine release in the stomach and small intestine during simulated digestion under fed conditions. Figure 6B shows the effect of capsule configuration on caffeine release in the stomach and small intestine during simulated digestion under fed conditions. Figure 6C shows the effect of capsule configuration on caffeine release in the stomach and small intestine during simulated digestion under fed conditions. Figure 6D shows the effect of capsule configuration on caffeine release in the stomach and small intestine during simulated digestion under fed conditions. [Figure 7] Figure 7A shows the effect of capsule configuration on caffeine release and probiotic survival. Figure 7B shows the effect of capsule configuration on caffeine release and probiotic survival. Figure 7C shows the effect of capsule configuration on caffeine release and probiotic survival. Figure 7D shows the effect of capsule configuration on caffeine release and probiotic survival. [Figure 8] Figure 8A shows the effect of capsule composition on the culturability of L. acidophilus strains after simulated digestion of the stomach and small intestine under fasted conditions, and Figure 8B shows the effect of capsule composition on the culturability of L. acidophilus strains after simulated digestion of the stomach and small intestine under fed conditions. [Figure 9] FIG. 1 shows the effect of probiotic administration through different capsules on microbial activity modulation in a simulated colonic environment.

[0019] definition As used herein, the terms "about," "approximately," or "generally," when used to modify a value, indicate that the value can be increased or decreased by 10% and still remain within the disclosed embodiment.

[0020] As used herein, the term "therapeutically effective amount" shall mean a dosage or amount of a composition administered or delivered to a mammal in need of such treatment that provides a particular pharmacological or nutritional response. It is emphasized that a "therapeutically effective amount" administered to a particular subject in a particular instance is not necessarily effective in treating a disease or otherwise improving health as described herein, but such a dosage is considered a "therapeutically effective amount" by those skilled in the art. Certain subjects may in fact be "refractory" to a "therapeutically effective amount". For example, refractory subjects may have low bioavailability or genetic variability in certain receptors, metabolic pathways, or response capabilities that do not result in clinical efficacy. It is further understood that the composition, or in certain cases supplements, may be measured as an oral dosage or in terms of component levels that can be measured in blood. In other embodiments, if the gut is the target of the active ingredient, the dosage may be measured in an amount that can positively affect the gut microbiome.

[0021] The term "nutraceutical" refers to any compound added to a dietary source (e.g., a food, beverage, or dietary supplement) that provides a health or medical benefit in addition to its basic nutritional value.

[0022] As used herein, the terms "delivering" or "administering" refer to any route for providing a composition, product, or dietary supplement to a subject that is accepted as standard by the medical community. For example, the present disclosure contemplates delivery or administration routes including oral ingestion.

[0023] As used herein, the term "mammal" includes any mammal that can benefit from improved joint health, resilience, mood, recovery, and general well-being, which may include, but is not limited to, canine, equine, feline, bovine, ovine, or porcine mammals. For purposes of this application, "mammal" includes human subjects and may be used interchangeably with animal.

[0024] As used herein, the term "capsule" refers to a conventional hard capsule or a gelatin capsule intended for oral administration to a mammal. A capsule has two coaxial telescopically joined parts, called a body and a cap. Usually, the cap and the body have a side wall, an open end, and a closed end. The length of each side wall of the parts is generally greater than the diameter of the capsule. The cap and the body of the capsule are telescopically joined together to overlap their side walls and obtain a capsule shell. "Partially overlapping" also encompasses an embodiment in which the side walls of the cap and the body have substantially the same length, such that the side walls of the cap envelop the entire side walls of the body when the cap and the body are telescopically joined. Thus, the capsule of the present invention does not structurally deviate from the conventional definition of a capsule. In general, "capsule" refers to both empty and filled capsules, while "shell" refers specifically to empty capsules. It is contemplated that when the hard capsule shell is filled with a substance in liquid form, the hard capsule of the present invention may be sealed or banded according to conventional techniques to avoid leakage of the contained substance.

[0025] As used herein, the term "acid resistance" or "acid-resistant" means that the capsule shells and capsules of the present invention exhibit no leakage for at least 1 hour when subjected to the USP disintegration test. For purposes of this disclosure, acid resistance is tested using the apparatus and procedures disclosed in the disintegration test for USP-30 dosage forms (essentially simulated gastric fluid TS at 37±2° C. in a basket / rack assembly).

[0026] The acid-resistant capsule shells and capsules of the present invention also exhibit satisfactory dissolution characteristics in simulated intestinal fluid at pH 6.8 and 37±2° C. in a paddle apparatus. Dissolution profiles of exemplary capsules of the present invention in simulated gastric and intestinal fluids are disclosed in the Examples and Figure 1. When tested in the dissolution test disclosed in the Japanese Pharmacopoeia 2 (JP2), the hard capsules of the present invention met the definition of an enteric-resistant hard capsule contained therein.

[0027] Other features and aspects of the disclosure are discussed in more detail below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] It should be understood by those skilled in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present disclosure.

[0029] The capsules usable in the present disclosure may be hard capsules. Both the inner capsule and the outer capsule may be hard capsules.

[0030] Suitable hard capsules include those that can be prepared from a capsule-forming aqueous composition that contains a film-forming polymeric matrix material, optionally one or more colorants, and water. Optionally, other additives such as plasticizers, antimicrobial agents, gelling agents, and neutralizing agents (especially alkaline materials) may be present.

[0031] The film-forming polymer matrix material may be selected from one or more celluloses, such as, for example, hydroxypropyl methylcellulose (HPMC or hypromellose), HPMCP, hydroxypropyl methylcellulose acetate succinate (HPMCAS), methylcellulose (MC); gelatin; pullulan; polyvinyl acetate or polyvinyl alcohol, and starch derivatives, such as hydroxypropyl starch and mixtures thereof, which form films with optimal mechanical performance in terms of elastic modulus and brittleness. In a particular embodiment, the film-forming polymer comprises HPMC and / or gelatin. In another embodiment, the film-forming polymer contains HPMC, which may be the only film-forming polymer matrix material. In a different embodiment, the film-forming polymer matrix material may contain gelatin as the only film-forming matrix material. Suitable types of HPMC are well known in the art, an example being HPMC type 2910 (as defined in USP30-NF25). Other types of HPMC are HPMC2208 and HPMC2906 (as defined in USP30-NF25). In one particular embodiment, the cellulose is hydroxypropyl methylcellulose (HPMC).

[0032] The HPMC methoxy and hydroxypropoxy content herein is expressed according to USP 30-NF 25. The viscosity of a 2 wt % aqueous solution of HPMC at 20° C. is measured according to the USP 30-NF 25 method for cellulose derivatives.

[0033] Typically, the aqueous composition comprises 10-50% by weight, more typically 15%-35% by weight, of the film-forming polymer, based on the total weight of the capsule-forming aqueous composition. Suitable hydroxypropyl methylcelluloses are commercially available. Generally, after forming the capsule and removing the water by drying, the film-forming polymer is the major component by weight of the final capsule shell. The use of water-soluble polymers in the dip-molding manufacturing process to form capsules is already known and has been widely disclosed in many publications and patents. The capsule formation process is described in more detail below. All water-soluble film-forming polymers currently used are commercially available.

[0034] In one particular embodiment, the HPMC in the aqueous compositions herein is HPMC having a viscosity of 4.0-5.0 cPs as a 2% w / w aqueous solution at 20° C. The viscosity of an aqueous HPMC solution can be measured by conventional techniques, for example, as disclosed in the USP, by using an Ubbelohde type viscometer. Suitable aqueous compositions can also be obtained by blending HPMC of the same type but different viscosity grades.

[0035] In one embodiment, the aqueous composition used to make the capsules herein may contain 0% to 5% by weight, typically 0% to 2% by weight, based on the total weight of the capsule-forming aqueous composition, of additional non-animal derived film-forming polymers typically used in the manufacture of hard capsules. In one embodiment, the HPMC aqueous composition of the present invention does not contain any film-forming polymers other than the presently disclosed HPMC. Non-animal derived film-forming polymers are, for example, polyvinyl alcohol, plant-derived or bacterial derived film-forming polymers. Exemplary plant-derived film-forming polymers are starch, starch derivatives, cellulose, cellulose derivatives, other than HPMC and mixtures thereof as defined herein. Exemplary bacterial derived film-forming polymers are exopolysaccharides. Exemplary exopolysaccharides are xanthan, acetan, gellan, welan, rhamsan, furcelleran, succinoglycan, scleroglycan, schizophyllan, tamarind gum, curdlan, pullulan, dextran, and mixtures thereof.

[0036] In one particular embodiment, the HPMC aqueous composition herein contains 0% to 1% by weight, preferably 0% by weight, based on the total weight of the capsule-forming aqueous composition, of an animal-derived material conventionally used in the manufacture of hard capsules. A typical animal-derived material is gelatin.

[0037] In another embodiment, the capsule-forming aqueous composition herein contains up to about 10% by weight of a gelling agent or gelling system, based on the total weight of the capsule-forming aqueous composition. Typically, the capsule-forming aqueous composition contains 0.2% to 5% by weight of a gelling agent or gelling system, based on the total weight of the capsule-forming aqueous composition. By "gelling system" is meant one or more cations with one or more gelling agents. Exemplary cations are K, + , Na + , Li + , NH4 + , Ca ++ , Mg ++, and mixtures thereof. Typical gelling agent(s) are hydrocolloids such as alginic acid, agar gum, guar gum, locust bean gum (carob), carrageenan, tara gum, gum arabic, ghatti gum, khaya grandifolia gum, tragacanth gum, karaya gum, pectin, arabian (araban), xanthan, gellan gum, konjac mannan, galactomannan, funoran, and mixtures thereof. As usual, the gelling agent can be used in combination with other ingredients, such as cations and sequestering agents, to form a gelling system. Commercially available capsules that can be used in the present invention include, for example, capsules available as VCaps® plus Color and PlantCaps® from Lonza Consumer Health Inc., Greenwood, South Carolina, USA.

[0038] In a different embodiment, depending on the film-forming polymer, the gelling agent or gelling system may be present in an amount of less than 0.2% by weight, based on the total weight of the capsule-forming aqueous composition, and for capsule-forming compositions essentially free of gelling agents or systems, typically less than 0.1% by weight, and even for capsule-forming compositions completely free of gelling systems, 0% by weight. If no gelling agent or gelling system is used, the film-forming polymer of the capsule-forming aqueous composition should be able to form a film without the need for a gelling agent.

[0039] In one particular embodiment, an aqueous HPMC composition containing average HPMC grade 2906 is suitable for obtaining strong and physically stable gels without a gelling system, and the dissolution properties of the HPMC capsules made therefrom are not adversely affected by the drawbacks typically associated with gelling systems, especially cationic. Capsules of this type are available from Lonza Consumer Health Inc., Greenwood, South Carolina, USA, under the trademark VCaps Plus®.

[0040] In another embodiment of the present disclosure, one of the capsules may be a delayed release capsule. Examples of delayed release capsules include capsules that are acid-resistant or enteric-coated. These capsules do not dissolve in the stomach or under acidic conditions, allowing the capsule contents to be delivered to the intestine of the user. Acid resistance can be achieved by coating a non-acid-resistant capsule with an enteric film. Enteric films include well-known acid-resistant materials that have pH-dependent water solubility. Typically, these materials are carboxylic acid group-containing polymers, such as cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), hydroxypropyl methylcellulose acetate succinate (HPMC-AS), acrylic copolymers, and shellac. These materials are water-insoluble under gastric conditions (conventionally simulated by pH 1.2) and readily water-soluble under intestinal conditions (conventionally simulated by pH 6.8). The disadvantages of coating solutions are typically represented by the complexity and expense of the manufacturing coating process, the high level of expertise required for its effective execution, the need to perform the coating at the end of the manufacturing cycle, i.e. after the capsules have already been filled, and finally, the need to contact the capsules with a solvent-based coating composition that may leave toxic solvent residues on the capsule surface after drying.

[0041] Therefore, other methods of achieving acid resistance or enteric properties have been developed. In one aspect, an acid resistant capsule can be prepared from an aqueous composition for the manufacture of an acid resistant pharmaceutical hard capsule, which comprises (i) an aqueous solvent, (ii) gellan gum, and (iii) one or more water soluble film forming polymers, characterized in that the weight ratio of gellan gum to the one or more water soluble film forming polymers is 4 / 100 to 15 / 100 (including lower and upper limits) by weight.

[0042] In a particular embodiment, the capsule-forming aqueous composition of the present invention contains (i) an aqueous solvent, (ii) gellan gum, and (iii) one or more water-soluble film-forming polymers, and the weight ratio of gellan gum to the one or more water-soluble film-forming polymers is 4 / 100 to 15 / 100, including lower and upper limits. The film-forming polymers for the acid-resistant or enteric capsules are the same as the film-forming polymers described above. These acid-resistant capsules are described in detail in U.S. Patent No. 8,852,631 to Cade et al., which is incorporated herein by reference.

[0043] The one or more water-soluble film-forming polymers contained in the capsule-forming aqueous composition are generally the major constituents by weight of the main component of the final capsule shell.The use of water-soluble polymers in the dip-molding manufacturing process for preparing delayed release or enteric hard capsules is already known and has been widely disclosed in many publications and patents.The water-soluble film-forming polymers currently used are all commercially available.

[0044] Gellan gum is an exopolysaccharide produced by fermentation. In the present invention, gellan gum is used in a ratio of about 4 to 15 parts by weight, preferably about 4.5 to 8 parts by weight, more preferably about 4.5 to 6 parts by weight (including lower and upper limits) per about 100 parts by weight of one or more water-soluble film-forming polymers. In a different embodiment of the present invention, gellan gum is used in a ratio of about 5 or 5.5 parts by weight per about 100 parts by weight of one or more water-soluble film-forming polymers. Based on experimental evidence, it is believed that if a lower amount of gellan gum is used, the final hard capsule does not have sufficient acid resistance under a disintegration test of pH 1.2, while a higher gellan content at process conditions (e.g., T and solids content) typical of conventional non-thermal gelling dip molding technology (for conventional processes, see, for example, the patent documents reported above) may cause excessive viscosity and excessive gelling capacity of the aqueous composition, thus making it impossible to manufacture capsules at the required high speed and quality. The preferred values ​​of gellan to polymer ratio are believed to provide an optimal combination of technical effects achieved by the present invention and processability aspects.

[0045] Due to its gelling properties, in contrast to gelatin, gellan gum is a typical component of the solidification system conventionally used in the manufacture of immediate release hard capsules when the water-soluble film-forming polymer used does not show satisfactory gelling properties by itself (e.g. HMPC or modified starch).However, in the prior art, gellan gum is typically used at a very low weight relative to the weight of the water-soluble film-forming polymer(s).For example, the amount of gellan gum typically used is less than 1 part by weight per about 100 parts by weight of the water-soluble film-forming polymer(s), which is significantly lower than the amount used in the present invention.

[0046] In addition, gellan gum is often used in combination with so-called gelling aids (typically salts of Na+, K+, or Ca2+). The use of small amounts of gellan gum and its combination with a gelling aid is taught to fully address the need to have the primary film-forming polymer gel on the dip pin to obtain a suitable hard capsule shell.

[0047] It has also been found that by working with the ratio of gellan gum to water-soluble film-forming polymer set forth above, suitable hard capsules can be obtained and acid resistance can be imparted to such capsules.

[0048] Another notable advantage is that the addition of so-called gelling aids is no longer necessary, even when working with film-forming polymers that have poor gelling properties themselves, such as HPMC. In other words, when gellan gum is used in the weight ratios indicated above, a composition suitable for the manufacture of hard capsules can be obtained from the HPMC or hydroxypropyl starch aqueous composition without adding gelling aids (e.g., cations) to the aqueous composition. The optional absence of the addition of gelling aids has an advantageous effect on the stability of the active ingredient filled in the final hard capsule shell, and on the hard capsule dissolution profile. The fact that the aqueous composition of the present invention does not contain the addition of gelling aids preferably means that it does not contain gelling aids, e.g., cations, in an amount not greater than the amount of the same aid naturally occurring in gellan gum. In another embodiment, the fact that the aqueous composition of the present invention does not contain the addition of gelling aids preferably means that it contains gelling aids, e.g., cations, in an amount not greater than the amount of the same aid naturally occurring in gellan gum. Such natural amounts can be readily established by routine laboratory testing on purchased batches of gellan gum, or it can be provided directly by a supplier of gellan gum.

[0049] The acid-resistant hard capsules do not leak in USP-30 simulated gastric fluid at pH 1.2 for at least 1 hour, confirming the acid resistance performance.

[0050] Typically, the total amount of components (ii) and (iii) (i.e., gellan combined with one or more water-soluble film-forming polymers) in the aqueous composition of the present invention is about 10% to 40% by weight, more preferably about 15% to 25% by weight, based on the total weight of the aqueous composition. Matching the appropriate concentration of film-forming polymer to the particular polymer used and the desired mechanical properties of the film is well within the capabilities of a person skilled in the art of hard capsule manufacturing. Commercially available delayed release capsules that can be used in the present invention include, for example, capsules available as VCaps®, DRcaps® from Lonza Consumer Health Inc., Greenwood, South Carolina, USA.

[0051] Optionally, the aqueous composition of the present invention may contain at least one inert, non-toxic pharmaceutical grade or food grade pigment, such as titanium dioxide, calcium carbonate iron oxide, and other colorants. Generally, 0.001 to 5.0% by weight of the pigment may be included in the aqueous composition. The weight is expressed over the total weight of solids in the aqueous composition.

[0052] Optionally, the aqueous composition of the present invention may contain a suitable plasticizer, such as glycerin or propylene glycol. To avoid excessive flexibility, the plasticizer content should be low, such as 0% to 20% by weight, more preferably 0% to 10% by weight, even more preferably 0% to 5% by weight, based on the total weight of solids in the aqueous composition.

[0053] Optionally, the aqueous compositions of the present invention may contain additional ingredients typically used in the manufacture of hard capsules, such as surfactants and flavoring agents, in amounts known to those skilled in the art and available in hard capsule publications and patents.

[0054] In another aspect, the present invention relates to an acid-resistant hard capsule shell obtained by using the aqueous composition defined above. In one particular embodiment, the shell comprises (I) moisture, (II) gellan gum, and (III) one or more water-soluble film-forming polymers, the weight ratio of gellan gum to the one or more water-soluble film-forming polymers being between 4 / 100 and 15 / 100 (including lower and upper limits).

[0055] In a preferred embodiment, the acid-resistant hard capsule shell is comprised of (I) moisture, (II) gellan gum, and (III) one or more water-soluble film-forming polymers, with the weight ratio of gellan gum to the one or more water-soluble film-forming polymers being between 4 / 100 and 15 / 100, inclusive. An exemplary commercially available capsule having acid resistance and delayed release of active ingredients is DRcaps® available from Lonza Consumer Health Inc., Greenwood, South Carolina, USA.

[0056] Whenever applicable, and unless technically incompatible, all features and preferred embodiments disclosed in relation to the aqueous composition of the present invention are also disclosed in relation to any other aspect of the present invention, including the acid-resistant hard capsule shell and the shell of the present invention.

[0057] The moisture content of the capsule shell of the present invention depends primarily on the water-soluble film-forming polymer or polymers used and on the relative humidity of the environment in which the shell is stored after production. Typically, the moisture content is about 2% to 16% by weight of the total weight of the shell. By way of example, under conditions conventionally employed for storage of hard capsules, the hard capsule shell of the present invention contains about 2 to 8% by weight, preferably about 2 to 6% by weight, preferably about 3 to 6% by weight of moisture by weight of the shell when the only film-forming polymer used is HPMC, and 10 to 16% by weight of moisture by weight of the shell when the only film-forming polymer used is gelatin.

[0058] In another aspect, the present invention relates to an acid-resistant hard capsule comprising a shell as defined above.

[0059] The capsules of the invention can be obtained by filling the shells of the invention with one or more substances to be encapsulated. After filling, the capsules can be made tamper-proof by making the seams durable, for example using suitable banding solutions used in the field of hard capsules.

[0060] In one particular embodiment, the hard capsule shell of the present invention as defined above is filled with one or more acid labile substances and / or one or more substances associated with gastric side effects in humans and / or animals.

[0061] In another aspect, the present invention relates to a dip-molding process for the production of acid-resistant pharmaceutical hard capsule shells, the process comprising: (a) immersing a pin in an aqueous composition as defined above; (b) withdrawing the dip pin from the aqueous composition; (c) drying the composition on the dip pin to obtain a shell; Steps (a)-(c) are performed in the order in which they are presented.

[0062] After the drying step (c), the resulting shell may be peeled off the pin and cut to the desired length. In this manner, a capsule shell part (body and cap) is obtained, which can then be elastically joined to form the final empty capsule. If filled with a liquid substance and if desired, after filling, the capsule can be made tamper-proof by suitable techniques known in the art, such as banding or sealing techniques, including those described in U.S. Pat. Nos. 9,579,290 and 9,980,918, and U.S. Patent Application No. 2020 / 0163893, each of which is incorporated herein by reference in its entirety.

[0063] Referring initially to FIG. 1, the dual capsule delivery device, generally indicated at 1, comprises a first, outer hard capsule 2 containing a liquid active ingredient 3, and a second, inner hard capsule 4, which also contains the same liquid active ingredient 5 as contained in the outer hard capsule 2, and may be coated as indicated at 6.

[0064] Similarly, Figure 2 shows a triple capsule delivery device, generally indicated at 11, which includes a first outer hard capsule 12 containing a liquid active ingredient 13, and a second inner hard capsule 14, which also contains the same liquid active ingredient 15 as contained in the outer hard capsule 12, and may be coated, as indicated at 18. The second inner hard capsule 14 also contains a third inner hard capsule 16, which in turn contains the same active ingredient as contained in the first outer hard capsule 12 and the second inner hard capsule 14, but in solid particulate form. The second inner hard capsule 14 may also be coated, as indicated at 19. Capsules 12, 14, and 16 are in series with one another.

[0065] 3, a multi-capsule delivery device, generally designated 21, includes a first outer hard capsule 22 containing a liquid active ingredient 23, and four inner hard capsules 24 which contain the same active ingredient 25 as contained in the first outer capsule 22, but in a semi-solid form. The four inner capsules 24 are parallel to one another but in series with the outer capsule 22.

[0066] Exemplary active substances that can be effectively delivered to a mammal include dietary supplements, pharmaceuticals, probiotics, and combinations thereof. The present disclosure is highly effective in allowing probiotics to survive passage through the stomach upon ingestion.

[0067] It has been shown that a capsule-in-capsule configuration can be used to deliver actives to the lower intestine of a mammal in need of treatment. It has been found that using an acid-resistant inner capsule within an outer capsule, whether the outer capsule is HPMC with gelling agent, heat-gelled HPMC, or gelatin or acid-resistant capsule, can be used in comparison to an acid-resistant capsule alone. It has been shown that in a heat-gelled outer capsule and an acid-resistant inner capsule, the amount of active ingredient delivered to the intestine, and particularly the lower intestine, can be increased by 10-fold, 20-fold, or even more. With an acid-resistant inner capsule and an outer capsule, the amount of active ingredient released can be 20-50-fold or more greater than a single acid-resistant capsule, regardless of whether the mammal is in a fasted or fed state.

[0068] The advantage of the present disclosure is a delivery system used to deliver active substances to mammals and provide benefits such as allowing high fermentation and consequently a significant increase in lactic acid production indicating functional probiotics, a significant decrease in propionic acid under fed conditions, indicating a decrease in propionic acid producing bacteria, an increase in butyric acid production under fasted conditions indicating an increase in butyric acid producing bacteria, and functional changes indicating a change in microbiome diversity in the gut. The capsule combinations suggested herein can be used to target release active substances to the intestines of the user and provide increased bioavailability of the active substances to the mammal.

[0069] Nonetheless, certain embodiments of the present disclosure may be better understood in accordance with the following examples, which are intended to be non-limiting and exemplary in nature. EXAMPLES

[0070] Example 1 Test procedure All reagents used in these examples were provided by Sigma (Overijse, Belgium) where otherwise stated.

[0071] Composition of the capsule system In this example, seven types of capsule-in-capsule systems and three single capsules were evaluated (Table 1). The capsule-in-capsule configurations were the following combinations of outer capsule (size number 00) and inner capsule (size number 3):

[0072] The capsules contained caffeine (50 mg / capsule) as a release marker and 2 × 10 10 A probiotic strain (L. acidophilus ATCC-43121, LGC Standards) was loaded at a concentration of CFU / capsule. [Table 1]

[0073] Capsule configuration testing Testing of the capsule formulation was completed in two phases. Phase 1 was an upper GIT sham study. Upon completion, phase 2 of the study was completed.

[0074] The upper GIT simulation experiments were carried out in two consecutive double jacketed reactors simulating gastric and small intestinal digestive conditions. The temperature was maintained at 37° C. and continuous magnetic stirring (300 rpm) was applied during the experiments. For capsule dissolution studies, the capsules were maintained in the gastric and small intestinal reactors using specially designed sinkers.

[0075] The pH profile, enzyme levels, and retention times were adjusted to mimic fed (i.e., consumption of the product during or immediately after a meal) and fasted (i.e., consumption of the product before a meal) conditions. Figure 4 shows the pH profile during the experiment under fed (A) and fasted (B) conditions. The pH of the medium was automatically controlled. The arrows indicate the start and end of the stomach (ST0, ST end) and the small intestine incubation phase. The difference is shown in Figure 4A and Figure 4B, where Figure 4A shows the pH profile for the fed condition and Figure 4B shows the pH profile for the fasted condition. As shown, the pH profiles during the experiment under fed (A) and fasted (B) conditions are different. ST = stomach, SI = small intestine. The pH of the medium was automatically controlled. The arrows indicate the start (ST0) and end (ST end) of the stomach and the small intestine incubation phase (start SI, end duodenum, end jejunum, and end ileum).

[0076] Under fasting conditions, gastric digestion was simulated by incubation for 45 min in gastric fluid (76 mL, pH 2) containing 0.66 g / L KCl, 3.63 g / L NaCl, and 3.95 g / L mucin, 0.4 mL lecithin (Carl Roth GmbH+Co.KG, Germany) (3.4 g / L), and 3.6 mL pepsin (Chem Lab, Zedelgem, Belgium) (10 g / L). Continuous pH control was performed by a Senseline pH meter F410 (ProSense, Oosterhout, The Netherlands) and an automated pump dosage of HCl (0.5 M) or NaOH (0.5 M) to keep the pH constant at 2. After gastric incubation, the gastric digestion volume was measured and adjusted to 100 mL with MilliQ water. The capsule sinker and gastric juice were transferred to the small intestine reactor and 35.2 mL of pancreatic juice (2.6 g / L NaHCO3, 4.8 g / L ox bile, and 1.9 g / L pancreatin), 2.15 mL of trypsin (10 g / L), and 2.7 mL of chymotrypsin (10 g / L) were added. The small intestine pH was gradually increased from 2 to 6.5 and maintained at this pH for 27 min, simulating duodenal incubation. Following this phase, the pH was increased stepwise (0.1 pH units every 7 min) to 7.5 within 63 min, simulating the jejunal environment. Finally, the pH remained constant at 7.5 during the 90 min ileal incubation. The increase in pH was achieved by the addition of NaHCO3 (8.4 g / L) at 60, 90, and 120 min, simulating dilution of the intestinal contents.

[0077] Under fed conditions, the study was performed in a similar manner to the fasted conditions, with the following modifications: Gastric digestion was simulated by incubation for 120 min in 76 mL of gastric juice solution containing SHIME® nutrient medium (20.53 g / L PDNM001B, ProDigest, Ghent, Belgium), NaCl (3.63 g / L), KCl (0.65 g / L), 0.4 mL lecithin (13.5 g / L), and 3.6 mL pepsin (40 g / L) at pH 4.6. During fed gastric digestion, a sigmoidal decrease in pH from 4.6 to 2 was obtained by controlled pumping of HCl (0.5 M) at established time points. After gastric incubation, the small intestinal phase was performed as described above but with a different composition of pancreatic juice (7.7 g / L NaHCO3, 15 g / L ox bile, and 10 g / L pancreatin), 2.15 mL trypsin (10 g / L), and 2.7 mL chymotrypsin (10 g / L). An increase in pH was achieved by addition of NaHCO3 (4.8 g / L) at 60, 90, and 120 min.

[0078] A blank control capsule with no caffeine or L. acidophilus was included in all assays as background medium for the caffeine HPLC analysis. Negative controls consisted of L. acidophilus and caffeine used alone without capsules. All assays were performed in triplicate.

[0079] Whole gastrointestinal simulation and colonic fermentation In the second phase of the study, the entire GIT study was completed. Following upper GIT incubation under the above fed and fasted conditions, colonic incubation was simulated by adding 160 mL of fresh colonic anaerobic medium [KH2PO4 (6.6 g / L), K2HPO4 (20.5 g / L), NaCl (5 g / L), yeast extract (2 g / L), peptone (2 g / L), glucose (1 g / L), starch (2 g / L), mucin (1 g / L), L-cysteine ​​HCl (0.5 g / L), Tween® 80 (2 mL)], 40 mL of anaerobic PBS [K2HPO4 (8.8 g / L), KH2PO4 (6.4 g / L), NaCl (8.5 g / L), and L-cysteine ​​HCl (0.5 g / L)]. A fixed pH interval of 6.5 to 5.8 was implemented and adjusted automatically by adding HCl (0.5 M) or NaOH (0.5 M). Colon incubations were then inoculated using fecal inocula derived from healthy donors as previously described.

[0080] Briefly, a 1:10 (w / v) mixture of fecal samples and anaerobic phosphate buffer (8.8 g / L K2HPO4; 6.8 g / L KH2PO4; 0.1 g / L sodium thioglycolate; 0.015 g / L sodium dithionite) was homogenized for 10 min (BagMixer 400, Interscience, Louvain-La-Neuve, Belgium). After centrifugation (2 min, 500 g) (Centrifuge 5417C, Eppendorf, VWR, Belgium) to remove large particles, fecal inoculum was added at 20% (v / v) to the upper GIT digesta of the different reactors. Colonic incubations were performed under anaerobic conditions at 37 °C and agitation at 90 rpm for 24 h (MaxQ 4000 Benchtop Orbital Shaker, Thermo Fisher Scientific, Belgium).

[0081] Quantification of caffeine release Caffeine was quantified by HPLC-UV / Vis (Hitachi Chromaster HPLC-DAD, Hitachi High-Tech Corporation, Japan) using an isocratic separation method (25% methanol:75% water) on a Kinetex® C18 LC column (serial number 00D-4601-E0, 5 μm, 100 Å, LC column 100×4.6 mm, core-shell silica solid support) (Phenomenex, Belgium). The column temperature was controlled and kept at 25±0.1° C. The total run time per sample was 7 min. The injection volume was 10 μL and the UV / Vis detector was operated at 272 nm. Caffeine quantification was performed using an external standard (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Samples were centrifuged at 5000 g for 15 min before injection onto the column. The supernatant was then filtered through a 0.2 μm filter into an HPLC vial. Caffeine analysis was performed on stomach samples at 15, 30, and 45 min (fed and fasted) and 60, 90, and 120 min (fed). Small intestine samples were collected at 30, 60, 90, 120, 150, and 180 min. Colon samples were obtained at 1, 2, and 24 h of incubation.

[0082] Survival of L. acidophilus by PMA-based qPCR Bacterial survival was tested by propidium monoazide (PMA)-based qPCR. For this procedure, a 1:1 (v / v) dilution of the sample in anaerobic phosphate buffer was mixed with 1.25 μL of PMAxx™ dye (20 mM) (VWR International Europe, Leuven, Belgium). The sample was incubated for 5 min in the dark under constant shaking (500 rpm) and centrifuged for 30 s at maximum speed (18.327 g). Afterwards, the sample was placed in the LED-active Blue system (GenIUL, Barcelona, ​​Spain), PhAST blue PhotActivation System (GenIUL, Barcelona, ​​Spain) for 15 min and centrifuged at 13.000 g for 10 min. The supernatant was immediately removed and DNA was isolated as previously described. qPCR was performed using primers specific for Lactobacillus acidophilus [L. acid_F (5'-GAAAGAGCCCAAACCAAGTGATT-3') and L. acid_R (5'-CTTCCCAGATAATTCAACTATCGC-3')] using a QuantStudio 5 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with program conditions previously described in (Van den Abbeele, Kamil et al. 2018). Survival of L. acidophilus was tested at the end of gastric incubation (45 min in fasted conditions and 120 min in fed conditions), 60, 120, and 180 min of small intestinal digestion, and 1, 2, and 24 h of colonic fermentation.

[0083] Cultivability of L. acidophilus The culturability of L. acidophilus was tested through MRS agar plates in samples obtained during the entire gastrointestinal transit. Samples were collected at the end of the stomach (45 min fasted and 120 min fed) and small intestinal phase (180 min) and serial 10-fold dilutions in anaerobic phosphate buffered saline were plated on MRS agar plates. Plates were incubated aerobically at 37°C for at least 48 h. The number of colony forming units (CFU) is reported as the mean log(CFU)±SEM (n=3).

[0084] Assessment of L. acidophilus functionality and metabolic activity of gut microbiota under colonic conditions During the 24-h colonic incubation, samples were obtained at 0, 1, 2, and 24 h for microbial activity assessment. pH measurements were performed using a Senseline pH meter F410 (ProSense, Oosterhout, The Netherlands). Short-chain fatty acids (SCFAs) (acetic acid, propionic acid, and butyric acid) and branched-chain fatty acids (BCFAs) (isobutyric acid, isovaleric acid, and isocaproic acid) were determined by gas chromatography as previously described (Ghyselinck, Verstrepen et al. 2020). Lactic acid production was assessed with a kit (R-Biopharm, Darmstadt, Germany) according to the manufacturer's instructions.

[0085] statistical methods Results are presented as mean and standard error of the mean (SEM) from triplicates. For multiple comparisons, a two-way ANOVA test including time and different conditions was applied, with t-Tukey's test. Statistical differences were set as p<0.05. Analysis was performed using GraphPad Prism software, version 9.0 (GraphPad Software, CA, USA).

[0086] result Characterization of capsule release behavior during upper gastrointestinal transit under fed and fasted conditions

[0087] In the first part of the study, ten capsule configurations shown in Table 1 were passed through a simulated upper GIT under fasted and fed conditions. Capsule dissolution was assessed at different time points in the stomach and small intestine digestive-like environment using caffeine as an activity marker.

[0088] Figure 5 shows the effect of capsule configuration on caffeine released during fasting conditions. Figure 5, left panel, shows caffeine release in the stomach. After 15 minutes of gastric digestion, there was a release of caffeine from Comparative Sample C3 (19.7 ± 1.3 mg) and to a lesser extent in Comparative Sample C2 (0.7 ± 0.3 mg) and Comparative Sample C1 (0.2 ± 0.04 mg). After 30 minutes of incubation, free caffeine increased rapidly for Comparative Sample C3 (40.8 ± 2.6 mg) and to a lesser extent in Comparative Sample C2 (5.7 ± 1.4 mg), Comparative Sample C1 (0.7 ± 0.1 mg). Sample E (0.5 ± 0.1 mg), Sample A (0.1 ± 0.003 mg), and Sample G (0.1 ± 0.02 mg). At the end of the gastric incubation (45 min), comparative sample C3 had the highest caffeine release (41.9±2.8 mg), indicating complete dissolution of the capsule. The other capsules showed partial caffeine release, with values ​​of 11.3±2.2 mg for comparative sample 2, 2.9±1.5 mg for sample E, and 1.5±0.3 mg for comparative sample 1. Finally, low caffeine release values ​​(0.1-0.2 mg), indicating high capsule integrity, were found for samples G, F, A, D, C, and B.

[0089] The right panel of Figure 5 shows the caffeine release in the intestine under the simulated digestive conditions described above. At the end of the duodenal incubation, there was a large increase in caffeine release for Comparative Sample C2 (36.4 ± 3.9 mg) and Sample E (27.0 ± 9.8 mg), and a small but constant caffeine release for Comparative Sample C1 (6.5 ± 1.5 mg), Sample D (5.8 ± 1.4 mg), Sample B (3.8 ± 0.6 mg), Sample G (1.7 ± 0.5 mg), Sample F (1.0 ± 0.1 mg), and Sample A (0.5 ± 0.2 mg). The Sample C capsule remained intact (0.2 ± 0.1 mg), but caffeine from Comparative Sample C3 had already been released during gastric incubation. After 60 minutes of small intestinal incubation, there was a large increase in caffeine release for sample D (42.6±4.8 mg) and sample E (36.5±3.0 mg), indicating complete dissolution of the capsule during the jejunal phase. Comparative sample C2 also disintegrated completely (41.8±2.3 mg). A slow but constant release continued for sample G (18.5±13 mg), sample B (14.3±1.2 mg), comparative sample C1 (12.7±1.7 mg), and sample F (3.8±0.1 mg), and sample A (2.5±0.9 mg). The first release of caffeine was detected for sample C in the jejunal incubation (0.9±0.2 mg). At the beginning of the ileal phase, after 90 minutes of small intestinal incubation, there was a large increase in caffeine release for sample B (38.2±4.6 mg) and sample G (31.1±9.8 mg), with more moderate values ​​for comparative sample C1 (20.1±3.4 mg), sample A (8.8±3.8 mg), sample F (8.8±0.1 mg), and sample C (30.1±0.7 mg). Further in the ileal phase, after 120 minutes of small intestinal incubation, samples B and G were completely dissolved. A large increase in caffeine release (31.6±6.6 mg) was observed for the sample A capsule. The other capsules, namely comparative sample C1 (26.2±4.7 mg), sample F (21.4±0.6 mg), and sample C (7±1.7 mg), still showed high integrity with a low and continuous caffeine release until the end of the small intestinal phase.At the end of the incubation, the capsules that were partially dissolved were sample C (24.2±6.8 mg), whereas sample F (46.3±4.2 mg), sample A (41.5±0.4 mg), and comparative sample C1 (36.7±6.5 mg) were completely dissolved.

[0090] FIG. 6 shows the effect of capsule configuration on caffeine released during the above feeding conditions. The left panel of FIG. 6 shows the results of caffeine release in the stomach under fed conditions. During feeding incubation (FIG. 6, left panel) and after 15 minutes of gastric digestion, caffeine was detected in Comparative Sample C3 (19.5±7.6 mg) and Comparative Sample C2 (2.4±1.4 mg), while after 30 minutes of gastric digestion, only small amounts of caffeine release (0.1-0.9 mg) occurred for Sample E, Comparative Sample C1, Sample A, Sample D, Sample F, and Sample G. A high increase in caffeine was observed for Comparative Sample C2 (32.2±3.3 mg) and Comparative Sample C3 (35.3±2.5 mg). After 45 minutes, the Comparative Sample C3 capsule dissolved. The Comparative Sample C2 capsule released 35.4±2.3 mg. The other capsules, Sample E (1.3±0.7 mg), Comparative Sample C1 (1.3±0.1 mg), Sample A (0.5±0.1 mg), Sample D (0.4±0.1 mg), Sample F (0.2±0.01 mg), and Sample G (0.2±0.1 mg), showed a slow but steady release, while Samples B and C showed the first signs of caffeine release (0.1±0.003 mg).

[0091] During the gastric incubation (60 min), the comparative sample C2 capsules are completely dissolved. A slow but constant release continues for the following capsules: sample D (5.2±2.3 mg), sample E (4.3±1.0 mg), sample A (1.9±0.1 mg), comparative sample C1 (2.3±0.4 mg), sample B (0.5±0.1 mg), sample G (0.5±0.2 mg), sample F (0.4±0.1 mg), and sample C (0.3±0.2 mg). After 90 min of gastric incubation, a large increase in caffeine release (39.8±0.1 mg) occurs for sample D (indicating complete dissolution of the capsule) and to a lesser extent in sample E (20.1±2.1 mg) and sample A (11.7±3.5 mg). A slow but steady release continued for Comparative Sample C1 (5.6±1.1 mg), Sample B (2.8±0.6 mg), Sample G (2.1±0.8 mg), Sample F (1.7±0.5 mg), and Sample C (0.2±0.01 mg). At the end of the gastric incubation, the Sample E capsule was completely dissolved. The other capsules were partially dissolved: Sample A (20.6±3.6 mg), Comparative Sample C1 (9.5±2.1 mg), Sample B (6.2±1.0 mg), Sample G (5.8±1.5 mg), Sample F (4.7±1.4 mg), and Sample C (0.7±0.04 mg).

[0092] The right panel of Figure 6 shows the results of caffeine release in the small intestine under fed conditions. Small intestinal incubation was started with the following four completely dissolved capsules: Comparative Sample C3, Sample D, Sample E, and Sample B. After duodenal incubation, the Sample A capsule was also completely dissolved (41.4 ± 0.7 mg). Moderate caffeine release continued in the small intestine for Sample G (23.7 ± 7.3 mg), Sample F (22.4 ± 4.8 mg), Comparative Sample C1 (14.5 ± 3.1 mg), Sample B (12.3 ± 1.9 mg), and Sample C (5.5 ± 0.1 mg). In the jejunal phase (60 min small intestinal digestion), caffeine release was complete for sample G (37.1 ± 5.0 mg) and sample F (35.7 ± 5.9 mg), while comparative sample C1 (18.8 ± 3.1 mg), sample B (17.7 ± 3.9 mg), and sample C (6.3 ± 3.8 mg) showed higher completeness to the digestion medium and lower caffeine release. In the ileal phase (90 min small intestinal digestion), sample G and sample F were completely dissolved. At the beginning of the ileal phase, there was a large increase in caffeine release for sample B (37.4 ± 7.8 mg), indicating complete dissolution of the capsule. Comparative Sample 1 (22.8±3.1 mg) and Sample C (15.8±3.2 mg) continued their moderate caffeine release until 120 minutes of small intestinal incubation, at which point all of the caffeine contained in Sample C was present in the digestive fluids (44.5±2.0 mg), indicating complete disintegration of the capsule. Only Comparative Sample C1 continued their moderate and constant caffeine release throughout further incubation until 32.7±2.5 mg of caffeine was finally released at the end of the small intestinal incubation. Based on the findings of the Phase 1 study, a Phase 2 study was completed with Sample B, Sample C, and Comparative Sam.

[0093] Protection of L. acidophilus by Sample C and Sample B during gastric and small intestine-like environment digestion promotes survival of the probiotic at the colonic level.

[0094] In the Phase 2 study, three capsule configurations (Sample C, Sample B, and Comparative Sample C3) were selected based on their delayed release in the Part 1 study to evaluate their behavior in the complete gastrointestinal tract under fasted or fed conditions. The survival of L. acidophilus in the colonic ecosystem and its regulatory effect were further tested. Comparative Sample C3 was selected because it was the most immediate release capsule and could be used as a control. Sample C was selected because it was the capsule-in-capsule configuration with the most delayed release of caffeine in the upper GI tract under fasted and fed conditions. The third capsule, Sample B, was selected as the second most delayed capsule-in-capsule configuration under fed conditions.

[0095] The effect of capsule configuration on caffeine release and probiotic survival is shown in Figure 7. Figures 7A and 7C show the time course of caffeine release during gastric, small intestinal, and colonic digestion. Figure 7A is fasted and Figure 7C is fed. The dots represent the caffeine content in the corresponding digestion or fermentation medium at the selected time points (mean ± standard error, n = 3). Figures 7B and 7D show the time course of Lactobacillus acidophilus survival during gastric, small intestinal, and colonic digestion. Figure 7B is fasted and Figure 7D is fed. The dots represent the copies / mL in logarithmic units of PMA-treated samples in the corresponding digestion or fermentation medium at the selected time points (mean ± standard error, n = 3).

[0096] As previously observed, in fasted conditions, caffeine release was significantly faster in comparative sample C3 than in the dual formulation (Figure 7A), indicating capsule disintegration before reaching the colonic environment. At the end of the small intestinal incubation period, sample C was partially dissolved, with the capsule completely dissolved after 1 hour of colonic incubation.

[0097] At the end of gastric incubation, the PMA-DNA copies of L. acidophilus (Figure 7B) were similar in both sample B (log 5.2 ± 0.1 copies / mL) and sample C (log 5.0 ± 0.2 copies / mL), while a higher PMA-DNA was detected in comparative sample C3 (log 7.94 copies / mL), likely due to a higher release of the probiotic strains into the digestive juices. However, after 60 min of small intestinal incubation, this number was reduced to log 6.2 ± 0.3 copies / mL, whereas for the other capsules, the PMA-DNA copies remained within similar values. After 120 min of small intestinal incubation, the PMA-DNA copies of L. acidophilus were log 8.8 ± 0.7 copies / mL in sample B and log 7.4 ± 1.2 copies / mL in sample C, indicating a high survival of the strains until the end of the small intestinal conditions.

[0098] The survival of L. acidophilus based on its growth on agar plates after gastric and intestinal passage is presented in Figure 8. Figure 8 shows the effect of capsule configuration on the culturability of L. acidophilus strains after simulated gastric and small intestinal digestion under fasted (A) and fed (B) conditions. Bars represent CFU in log units (mean ± standard error, n = 3) obtained by plate counting from gastric and small intestinal digesta exposed to different capsule configurations. Product refers to the maximum L. acidophilus CFU inoculated in the different capsules. Significant differences are marked with asterisks (p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ***). L. acidophilus from Samples C and B showed significantly higher growth than when it was included in Comparative Sample C3 under fasting conditions in the small intestinal environment, but in the fed state, differences were observed in the gastric phase after 120 min, with colony forming units (CFU) in Samples C and B being more moderate than in Comparative Sample C3. This is likely caused by a higher release of capsule contents from Comparative Sample C3 in the gastric medium.

[0099] In the simulated colonic environment, the effect of probiotic administration on microbial activity through the three capsules was measured at different time points (Figure 9). In general, the effects observed under fasting conditions were less than under fed conditions. Butyrate was the most affected metabolite.

[0100] Under fasting conditions, the only significant difference was observed between sample B and comparative sample C3 (Figure 9). Notably, butyric acid was increased when L. acidophilus supplementation was included in sample C (6.0 ± 0.3 mM) and sample B (5.6 ± 0.3 mM) compared to the control sample C3 (3.4 ± 0.1 mM). Ammonium levels were slightly increased in comparative sample C3 (156.1 ± 6.1 mg / L) compared to sample B (143.8 ± 1.7 mg / L), while BCFAs showed the opposite trend and were significantly decreased in comparative sample C3 (0.3 ± 0.01 mM) compared to samples C and B (0.48-0.5 mM).

[0101] Under fed conditions, the pH reduction was higher in sample C and sample B (-0.6 ± 0.01 Δ24-0 h), but lactate levels increased significantly in both dual configurations (1.3-2.8 mM). In particular, acetate and propionate were reduced in sample C (acetate = 35.0 ± 0.8 mM, propionate = 7.4 ± 0.01) compared to the other conditions (acetate = 38.2-42.5 mM, propionate = 8-9.1 mM). In contrast, the highest butyrate levels were detected in the reactors of sample B (6.6 ± 0.3 mM), with the opposite effect observed for ammonium (108.8 ± 4.2 mg / L). Under fed conditions, there were no significant differences in branched chain fatty acid production between the different capsules.

[0102] Discussion of results Targeted delivery of pharma- ceutical active compounds, nutritional supplements, or probiotics is essential for product performance as well as probiotic viability and its functions, including colonization and microbiome modulation.

[0103] The most common capsule material has been gelatin due to its convenience, low cost, non-toxicity, solubility in biological fluids at body temperature, and gelling characteristics. However, several drawbacks have been described for gelatin, such as reactivity to aldehyde groups, sugars, metal ions, plasticizers, or preservatives. In addition, high environmental humidity, dependent temperature release, and moisture changes due to animal (porcine) origin are all disadvantages of gelatin. HPMC is a plant-based material, has low cross-reactivity with excipients, is stable over a wide range of temperature and moisture conditions, and has proven safety for human consumption, so it meets multiple criteria to replace gelatin-based capsules.

[0104] The aim of this study was to evaluate the release and disintegration characteristics of different HPMC-based capsule combinations in a capsule-in-capsule configuration, using caffeine bioavailability and probiotic survival as markers. The SHIME model is used to simulate full length gastrointestinal tract conditions. We found that the combination with comparative sample C1 showed delayed caffeine release in the stomach and small intestine under both fed and fasted conditions, and conferred a significant increase in probiotic survival and performance at the colon level.

[0105] The nature and concentration of the gelling agent determine the release behavior. Our study showed that at the end of the fasted and fed gastric environment, the caffeine release was complete in the single Comparative Sample C2 capsule, while the profile was lower in Comparative Sample C1. Both Comparative Samples C2 and C1 are made from HPMC, with a gelling agent (gellan gum) incorporated in Comparative Sample C1 compared to Comparative Sample C2. The insolubility of gellan at pH below 4 and the change in the physical properties of the HPMC film due to gelation may increase the resistance to mechanical stress during gastric passage and may be responsible for the delayed release behavior of Comparative Sample C1. It has been reported elsewhere that HPMC capsules containing carrageenan as a gelling agent showed a fast disintegration profile in vivo under fasted conditions (complete release after 7-9 minutes), similar to gelatin capsules. In addition, due to the lower mechanical strength of the cellulose film, a gelling additive is also required for the capsule shell HPMC production. Carrageenan and potassium chloride have proven effective in HPMC gelling, while gellan gum in combination with ethylenediaminetetraacetic acid (EDTA) or sodium citrate has been used in HPMC capsule manufacturing.

[0106] In the small intestinal phase, the highest delayed caffeine release was observed for sample C under fasting conditions and for comparative sample C1 under fed conditions, but neither achieved full caffeine delivery even at the distal part of the small intestine. This observation suggests that sample C can be used to deliver colon-targeted probiotics that are viable at their site of action. Probiotic viability along with storage or administration is a key factor in their efficacy. Thus, survival of orally administered probiotics is necessary for their performance.

[0107] Caffeine release from comparative sample C1 followed a linear trend (R2>0.9) under both fed and fasted conditions, suggesting a time-sustained steady-state delivery, which may also be beneficial for the engraftment of probiotics in the gut. The changes in the SCFA profile suggest that other bacteria from the microbiota are affected by the introduction of exogenous L. acidophilus, indicating that this targeted delivery to the colon allowed for modulation of the microbiome. In particular, the observed increase in lactate suggests colonization by L. acidophilus. A sufficient mass of viable "engrafted" microorganisms introduced into a complex ecosystem can outcompete other commensal organisms, thus modulating the diversity of the microbiome. This process is known as the growth pressure hypothesis, whereby a successful invasion requires a sufficient number of individuals to enter the ecosystem, which is related to the cell number (or dose) of the treatment and the frequency with which they are applied. Due to the resilience of the pre-established microenvironment of commensal microorganisms, probiotic strains do not easily engraft in the human gut ecosystem. However, under conditions of gut dysbiosis imbalance, for example after antibiotic intake, the potential benefits of probiotic microorganisms to colonize and restore gut homeostasis can be improved by targeted colonic delivery using a capsule-in-capsule configuration. Indeed, previous in vivo studies have shown that acid-resistant capsules in a capsule-in-capsule configuration are resistant to the low pH gastric environment under fasting conditions. The same authors report high inter-individual variability in gastric emptying times, which may significantly affect disintegration times and product release. Despite in vivo conditions that may differ from in vitro tests due to the complex nature and inter-individual variability of gastrointestinal processes, different in vitro models simulating gastrointestinal digestion have been developed to mimic human physiology under fasting and fed conditions. Physiological gastric and intestinal pH and bile salt concentrations undergo gradual changes during the digestive process, which were reproduced in this study by constant addition of acid and digestive fluids, improving on previously developed static setups (including duodenal, jejunal, and ileal phases) at different pH, retention times, and bile salt concentrations, making the in vitro system more similar to gastrointestinal digestion in humans.

[0108] The changes in caffeine release were accompanied by differences in L. acidophilus viability, especially under fasting conditions. To further evaluate the function of L. acidophilus at its site of action, we evaluated whether these changes in probiotic viability had an effect on gut microbial regulation under colonic conditions. For three selected capsules, gastrointestinal digestion was continued with simulated colonic fermentation. Detection of viable L. acidophilus in the colonic environment was significantly higher when administered in sample C or sample B. Comparative sample C3 was used as a negative control, as suggested by the lactate reduction. In addition, sample C and sample B also influenced the colonic composition and diversity of microorganisms, based on the resulting reduction in acetate and propionate and increase in butyrate. The protection of L. acidophilus could potentially have induced higher colonic medium acidification and lactate production by providing lactate as a substrate for other bacteria in the microbiota (cross-feeding interaction). It has been previously described that probiotic Lactobacillus species can ferment non-digestible fiber to allow lactic acid production, which is then used as a substrate by butyrate-producing bacteria. Butyrate is a microbial metabolite that plays an important role in maintaining gut homeostasis, including immune regulation, intestinal motility, and epithelial barrier function. An increase in butyrate may reflect an increase in butyrate-producing bacteria. A decrease in acetate and propionate may reflect a decrease in SCFA-producing bacteria.

[0109] Low gastric pH and high bile acid concentration are the main factors that reduce the survival rate of probiotics. Therefore, delayed release formulations such as Comparative Sample C1 or Sample B targeted for colonic delivery can improve probiotic performance in regulating gut microbial diversity and composition, as observed in this study in vitro, which leads to various health benefits. On the other hand, the fast caffeine release from Comparative Sample C3 may suggest that this formulation can be used for targeted gastric release.

[0110] Example 2 MRI testing Various capsules in the capsule formulation were tested for efficacy. The capsules and configurations are shown in Table 2. [Table 2]

[0111] The capsules were filled with the powder mixture. The powder mixture used to fill the capsules consisted of common excipients known not to affect the disintegration behavior of the capsule. For the single capsules (study groups I, II, and VII), the filling mixture consisted of 5% black iron oxide, 12% croscarmellose, 10% 13C3-labeled caffeine (=25 mg), and standard capsule filling powder (99.5% mannitol and 0.5% silicon dioxide).

[0112] The capsule-in-capsule configurations (Groups III-VI and VIII-X) were filled with a similar mixture, but because the inner capsule was smaller, the amount of caffeine was higher on a percentage basis. The caffeine content of the delivery form was determined to be constant at 25 mg in each capsule. The inner capsule mixture also consisted of 5% black iron oxide, 12% croscarmellose, 23% 13C3 labeled caffeine (=25 mg), and standard capsule fill powder (99.5% mannitol and 0.5% silicon dioxide). The outer capsule fill was one of the smaller capsules (Size 3, depending on the study group) and the gap was filled with a mixture of 7.2% naturally occurring caffeine (=25 mg) and hibiscus tea powder. The amount of caffeine was approximately 50 mg per capsule (25 mg 13C3 labeled and 25 mg natural caffeine). All capsule types were filled manually on a laboratory scale to target fill weights of 250 mg for size 00 single capsules, 106 mg for size 3 capsules, and 300 mg for size 00 capsule-in-capsule combinations.

[0113] A healthy volunteer study was performed. It was conducted as an open-label, single-center, 10-way crossover study with at least 72 hours washout between study days. Six healthy young volunteers (2 males and 4 females) were recruited for the study. These subjects had a mean age of 23.2±3.6 years and a mean BMI of 23.5±2.6 kg / m2. Volunteers were required to abstain from caffeine-containing foods such as coffee, tea, and chocolate products for at least 3 days and throughout each study day.

[0114] Each capsule type was investigated as a single capsule (Groups I, II, and VII). In addition, seven different capsule combinations were investigated. For each study group, the observation period was set based on the estimated maximum disintegration time of the individual capsule or the inner capsule of the capsule-in-capsule configuration shown in Table 2.

[0115] Between study days there was a washout phase of at least 72 hours. All subjects arrived at the study unit in the morning after an overnight fast of at least 10 hours. For each study group, a fasting MRI was obtained at -5 minutes and a blank saliva probe was obtained at -2 minutes to ensure identical clinical conditions, respectively. Time 0 minutes was defined as the time when the capsule was taken in an upright position with 240 mL of water. All study groups consisted of a 60-minute observation period with 10-minute intervals, with an additional 120-minute observation period with 15-minute intervals added for study groups V-X, and a further 60-minute observation period with 15-minute intervals performed for study group X. At each observation time point, two MRI sequences (TRUFI and VIBE) were applied to be able to distinguish between the two contrasting agents, iron oxide and hibiscus tea powder. Sequence parameters are listed in Tables 3 and 4.

[0116] The T2* / T1 weighted TRUFI sequence is highly sensitive to magnetic susceptibility artifacts generated by magnetic materials such as applied ferrimagnetic black iron oxide. This characteristic artifact does not depend on the hydration state, and therefore we applied it to the detection of intact capsules. As soon as the capsules containing iron oxide disintegrate, the iron oxide diffuses, which is visible as a widening of the artifact. To accelerate the diffusion of powdered iron oxide, croscarmellose, a strong disintegrant, was added to the capsule filling mixture. In contrast, dry hibiscus tea powder is not visible in any order. However, as soon as it comes into contact with water, the contained paramagnetic elements such as manganese extend the T1 water proton signal, which can be detected as a bright spot in the VIBE sequence. The labeling of hibiscus tea powder was therefore aimed at the detection of the disintegration of the outer capsule. Nevertheless, the main aim of this study was to investigate the fate of the inner capsule, which is a vehicle relevant for clinical applications.

[0117] If complete capsule disintegration could not be observed within the scheduled observation time, the measurement was extended for 30 min (two additional measurements). For study groups with only single capsules (I, II, and VII), only the TRUFI sequence was performed because the single capsules did not contain the hibiscus tea powder that the VIBE sequence was aimed at. Saliva samples were always obtained 1 min after imaging.

[0118] MR imaging was performed at the Institute of Diagnostic Radiology and Neuroradiology in Greifswald using a Siemens MAGNETOM Aera MR-scanner (Siemens Healthcare, Erlangen, Germany) with a field strength of 1.5 Tesla. All measurements were performed in supine position (subject lying on his back, head facing forward). Two different spatial orientations (lateral and coronal) were used while the artifact was in the stomach, and the coronal orientation was used only after gastric emptying. [Table 3] [Table 4]

[0119] Image analysis Image analysis was performed using Horos Viewer Version 3.3.6. Tracking, assignment to gastrointestinal compartments, and assessment of disintegration time points were performed manually. All recordings were independently evaluated by three independent observers, and unclear findings were discussed.

[0120] The appearance of bright spots in the VIBE sequence caused by the moistened hibiscus tea powder was defined as the collapse of the outer capsule. In turn, the time point at which the collapse of the inner capsule was detected (detected in the TRUFI sequence) was defined as the time of diffusion of the characteristically shaped magnetic susceptibility artifact in the GI tract or the visible sedimentation of iron oxide in the stomach. The part of the GI tract where the artifact or the corresponding particle (hibiscus tea powder or black iron oxide) was located when the collapse was determined was scored as the site of collapse of the respective (outer or inner) capsule.

[0121] result With the exception of one administration of the combination in study group III and one administration of the combination in study group IV (because these study groups took longer to disintegrate than all other subjects and therefore longer than the planned MRI observation time), the localization of the capsules and their disintegration could be clearly identified in the TRUFI sequence. Their disintegration could not be detected within an additional 30 minutes of imaging. The findings obtained by MRI are summarized in Tables 5 and 6. The results show that it is possible to delay the overall disintegration time by placing a capsule inside another capsule. For some combinations, the disintegration time was almost exactly the sum of both disintegration times determined for the individual capsules, for example 23 minutes for the single study group I and 40 minutes for study group III. This was not the case when an acid-resistant capsule was included in the capsule-in-capsule configuration. Here, the disintegration time of the inner capsule was typically longer than the sum of the disintegration times of the outer and inner capsules. The increase in disintegration time was often accompanied by an increase in variability (±5 minutes and ±12 minutes for HPMC capsules with gelling agent and thermogelled HPMC compared to ±18 minutes for study group IV).

[0122] The site of disintegration of the single capsules and the capsule combinations were also highly variable (Table 5). Two exceptions were observed. The combinations of Study Group II and Study Group X disintegrated most reproducibly with their different properties. Thus, the combination of the two acid-resistant capsules led to disintegration in the ileum for all six doses. HPMC with gelling agent and thermogelled HPMC and their combinations showed short disintegration times leading to disintegration in the stomach or proximal parts of the small intestine, whereas the acid-resistant capsules and their combinations with an acid-resistant capsule as the outer shell disintegrated mainly in the small intestine. None of the combinations or single capsules tested reached the colon. The thermogelled HPMC capsules showed fast gastric disintegration with very low variability in disintegration time and site. In total, 4 of 24 doses with HPMC with a gelling agent capsule as the outer shell and 1 of 24 doses with an acid-resistant capsule as the outer shell disintegrated in the esophagus. None of the subjects noticed any adhesion of the capsule to the esophagus or described any negative sensations. [Table 5]

[0123] Consequences of the Caffeine Decision The results obtained by salivary caffeine determination are summarized in Table 6 together with the MRI results. The mean salivary 13C3-caffeine appearance times determined for the single size 00 capsules were 22±12 minutes for study group I, 15±0 minutes for study group II, and 25±11 minutes for study group VII. These times are in good agreement with the salivary caffeine appearance times determined for the capsule-in-capsule configurations using these capsules as the outer capsules. In agreement with the MRI results, the study group X combination showed the longest salivary caffeine appearance time at 115±31 minutes and the longest disintegration time at 123±25 minutes. Also as observed in the MRI, the thermally gelled HPMC capsules had the lowest variability in disintegration time and salivary caffeine appearance.

[0124] Table 6 shows the average disintegration times determined by MRI, as well as the saliva appearance times of natural caffeine and 13C3-labeled caffeine. In addition, the time span between the disintegration of the inner shell and the disintegration of the outer shell, and the gastric emptying time are given. In general, caffeine appears at or before the detection of disintegration by MRI. However, a trend towards later disintegration times and higher variability of the capsule combinations is evident for both, with the ratios of the study groups being very similar to each other. [Table 6]

[0125] These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art without departing from the spirit and scope of the present invention, which is particularly set forth in the appended claims. In addition, it should be understood that aspects of the various embodiments may be interchanged in whole or in part. Moreover, those skilled in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention as further set forth in such appended claims.

Claims

1. A delivery system for providing oral administration of an active substance to a mammal, wherein the delivery system comprises: a. an outer capsule having an outer shell wall and an internal chamber; b. an inner capsule having an outer shell wall and an inner compartment, wherein the inner capsule is located within the internal chamber of the outer capsule and the inner capsule is acid-resistant; c. an active substance, wherein the active substance is present within the inner compartment of the inner capsule; and the delivery system is orally administered to a mammal and delivers the active substance in an effective amount to the intestine of the mammal.

2. The delivery system according to claim 1, wherein the outer capsule comprises an HPMC hard capsule.

3. The delivery system according to claim 1, wherein the inner capsule comprises an HPMC hard capsule having acid resistance.

4. The delivery system according to claim 3, wherein the inner capsule comprises a capsule containing HPMC and gellan gum.

5. The delivery system according to claim 4, wherein the gellan gum is present in an amount of about 4 to 15 parts per 100 parts of the HPMC.

6. The delivery system according to claim 2, wherein the HPMC capsule comprises thermogelated HPMC.

7. The delivery system according to claim 1, wherein the outer capsule is an acid-resistant capsule.

8. The delivery system according to claim 7, wherein the outer capsule comprises an HPMC hard capsule having acid resistance.

9. The delivery system according to claim 8, wherein the outer capsule comprises a capsule containing HPMC and gellan gum.

10. The delivery system according to claim 9, wherein the gellan gum is present in an amount of about 4 to 15 parts per 100 parts of the HPMC.

11. The delivery system according to any one of claims 1 to 10, wherein the active substance comprises probiotics.

12. The delivery system according to claim 1, wherein the inner capsule and the outer capsule are each acid-resistant capsules, and each acid-resistant capsule contains HPMC and gellan gum.

13. The delivery system according to claim 12, wherein the gellan gum is present in an amount of about 4 to 15 parts per 100 parts of the HPMC.

14. The delivery system according to claim 1, wherein the inner capsule comprises two or more inner capsules.

15. The delivery system according to any one of claims 1 to 10, wherein the inner capsule is prohibited.

16. The delivery system according to any one of claims 1 to 10, wherein the active substance is delivered to the colon in an amount at least 10 times greater than that of a capsule that dissolves in the stomach or small intestine.

17. The delivery system according to claim 16, wherein the active substance is delivered to the colon in an amount at least 20 times greater than that of a capsule that dissolves in the stomach or small intestine.

18. The delivery system according to claim 17, wherein the active substance is delivered to the colon in an amount at least 30 times greater than that of a capsule that dissolves in the stomach or small intestine.

19. A delivery system for modifying the microbiome and colonization of the intestine of a mammal, wherein the delivery system comprises: a. an outer capsule having an outer shell wall and an internal chamber; b. an inner capsule having an outer shell wall and an inner compartment, wherein the inner capsule is located within the internal chamber of the outer capsule, and the inner capsule is acid-resistant; c. a probiotic active ingredient, wherein the active substance is present within the inner compartment of the inner capsule; and the delivery system is orally administered to a mammal, the delivery system delivers the probiotic active substance in an effective amount to the intestine of the mammal, and the active ingredient improves the microbiome or colonization of healthy bacteria in the intestine.

20. The delivery system according to claim 19, wherein the outer capsule comprises an HPMC hard capsule.

21. The delivery system according to claim 20, wherein the inner capsule comprises an HPMC hard capsule having acid resistance.

22. The delivery system according to claim 20, wherein the inner capsule comprises a capsule containing HPMC and gellan gum.

23. The delivery system according to claim 22, wherein the gellan gum is present in an amount of about 4 to 15 parts per 100 parts of the HPMC.

24. The delivery system according to claim 20, wherein the HPMC capsule comprises thermogelated HPMC.

25. The delivery system according to claim 19, wherein the outer capsule is an acid-resistant capsule.

26. The delivery system according to claim 25, wherein the outer capsule comprises an enteric HPMC hard capsule.

27. The delivery system according to claim 26, wherein the outer capsule comprises a capsule containing HPMC and gellan gum.

28. The delivery system according to claim 27, wherein the gellan gum is present in an amount of about 4 to 15 parts per 100 parts of the HPMC.

29. The delivery system according to claim 19, wherein the inner capsule and the outer capsule are each an enteric capsule, and each enteric capsule contains HPMC and gellan gum.

30. The delivery system according to claim 29, wherein the gellan gum is present in an amount of about 4 to 15 parts per 100 parts of the HPMC.

31. The delivery system according to claim 19, wherein the inner capsule comprises two or more inner capsules.

32. The delivery system according to any one of claims 19 to 31, wherein the inner capsule is prohibited or has a prohibited substance at the joint of each part of the capsule.

33. The delivery system according to any one of claims 19 to 31, wherein the active substance is delivered to the colon in an amount at least 10 times greater than that delivered by a capsule that dissolves in the stomach or small intestine.

34. The delivery system according to claim 33, wherein the active substance is delivered to the colon in an amount at least 20 times greater than that delivered by a capsule that dissolves in the stomach or small intestine.

35. The delivery system according to claim 34, wherein the active substance is delivered to the colon in an amount at least 30 times greater than that delivered by a capsule that dissolves in the stomach or small intestine.

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