Oral delivery system based on in situ formation of protein / polysaccharide aggregates

Abstract

This article describes an oral delivery system based on the in-situ formation of protein / polysaccharide aggregates. The system comprises an active ingredient dispersed in a dry, homogeneous powder mixture of protein and polysaccharide powders, which is capable of forming protein / polysaccharide complex aggregates in situ upon immersion in gastric fluid, thereby conferring gastric protection and / or improved release of the active ingredient. Changing the ratio of protein to polysaccharide powder in the oral delivery system alters the level of gastric protection and / or the release rate of the active ingredient.

Description

Technical Field

[0001] This specification relates to oral delivery systems for active ingredients. More specifically, this document describes a multifunctional solid oral delivery system comprising a protein / polysaccharide powder mixture that forms in situ protein / polysaccharide complex aggregates upon exposure to the gastric environment, thereby providing gastric protection and / or improved release of the active ingredient. Background Technology

[0002] Modified-release oral delivery systems are specific products formulated to allow for modulated release of the active ingredient. This allows for the achievement of specific goals that are not possible with conventional dosage forms. These products offer a variety of advantages, including potential therapeutic benefits, improved efficacy, reduced adverse effects, optimized performance, and increased convenience and patient compliance. While various modified-release oral delivery systems exist, many of these systems use synthetic compounds and / or polymers, which are increasingly being avoided by consumers (and therefore companies), particularly for active ingredients intended for regular administration. Furthermore, many currently used modified-release oral delivery systems require complex, multi-step manufacturing processes, such as applying multiple coatings or layers to the oral dosage form. Therefore, there is a strong need for alternative oral delivery systems that incorporate more natural ingredients and simplify manufacturing processes. Summary of the Invention

[0003] On one hand, this article describes an oral delivery system comprising a dry, homogeneous mixture containing a mixture of protein powder and polysaccharide powder, and an active ingredient dispersed therein. Upon immersion of the oral delivery system in gastric fluid, the protein and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ, thereby imparting gastric protection to the active ingredient and / or improved release of the active ingredient. The ratio of protein powder to polysaccharide powder in the oral delivery system is varied to alter the level of gastric protection of the active ingredient and / or the release rate of the active ingredient.

[0004] On the other hand, this article describes a method for preparing a solid oral dosage form, the method comprising dispersing an active ingredient in a dry, homogeneous mixture comprising protein powder and polysaccharide powder, and formulating the resulting mixture into a solid oral dosage form, wherein the polysaccharide powder has powder flow characteristics and is capable of interacting with the protein powder such that immersing the solid oral dosage form in gastric juice results in the in-situ formation of a protein / polysaccharide complex aggregate, thereby imparting gastric protection to the active ingredient and / or improved release of the active ingredient.

[0005] On the other hand, this document describes a method for in situ generation of protein / polysaccharide complex aggregates that impart gastric protection to an active ingredient dispersed therein and / or improved release of the active ingredient, the method comprising providing the active ingredient formulated in an oral delivery system according to the present invention or in an oral dosage form produced according to the method described herein, and orally administering the oral delivery system or the oral dosage form to a subject, wherein the protein and polysaccharide powder mixture forms the protein / polysaccharide complex aggregate in situ after the oral delivery system is immersed in the gastric fluid of the subject.

[0006] On the other hand, this article describes a method for treating a disease or condition that is improved by administering to a subject an active ingredient that will benefit from gastric protection and / or improved release. The method comprises providing the active ingredient formulated in an oral delivery system according to the present invention or in an oral dosage form produced according to the method described herein, and orally administering the oral delivery system or the oral dosage form to a subject, wherein, after immersing the oral delivery system in the gastric fluid of the subject, the protein and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ.

[0007] In implementation, the polysaccharide powder suitable for the oral delivery system described herein has specific powder flow characteristics associated with powders that have increased cohesion (or decreased flowability), characterized by parameters such as the angle of repose (α), dynamic cohesion index, Hausner ratio, and / or minimum thresholds of compressibility index (Carr index).

[0008] General definition

[0009] The headings and other identifiers presented, such as (a), (b), (i), (ii), etc., are for ease of reading the specification and claims only. The use of headings or other identifiers in the specification or claims does not necessarily require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0010] The use of the word "a" or "an" in conjunction with the term "comprising" in the claims and / or specification may mean "a," but it is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0011] The term "about" is used to indicate that a value includes the standard deviation of the error of the apparatus or method used to determine that value. Generally, the term "about" means a possible variation of up to 10%. Therefore, values ​​of variation of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% are included in the term "about". Unless otherwise stated, the use of the term "about" before a range applies to both ends of the range.

[0012] As used in this specification and one or more claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unlisted elements or process / method steps. Attached Figure Description

[0013] In the attached diagram:

[0014] Figure 1 In vitro release curves of formulations 2-1 to 2-7.

[0015] Figure 2 Typical stress-strain curve (simulating formulation 7 in gastric juice [SGF])

[0016] Figure 3 The deconvolution amide I' bands were obtained from formulations 2-5, 2-6, and 2-7 after 2 hours at pH 1.0 (SGF) or pH 6.9 (SIF).

[0017] Figure 4 According to the United States Pharmacopeia (USP) <711> The standard is to ensure the survival of Pediococcus acidilactici after immersing formulations 4-1, 4-2 and 4-3 in simulated gastric conditions for 1 or 2 hours.

[0018] Figure 5 The remaining capsule content after immersion of formulation 4-3 in SGF for 2 hours: The protein / polysaccharide mixture forms a complex aggregate suitable for capsule shape.

[0019] Figure 6 Caffeine release curves obtained from the formulations described in Table 4.

[0020] Figure 7 : Schematic diagram of the process of capsule dissolution and aggregate formation complex.

[0021] Figure 8A Postgastric survival rate of probiotic strains obtained from the formulations described in Table 13.

[0022] Figure 8B Survival rate of individual probiotic strains (unformulated).

[0023] Figure 9 The disintegration kinetics of the formulations described in Table 13.

[0024] Figure 10 Dissolution profiles obtained from the formulations described in Table 14.

[0025] Figure 11 Dissolution profiles obtained from the formulations described in Table 15.

[0026] Figure 12 The dissolution characteristics of the formulations described in Table 16.

[0027] Figure 13 Dissolution characteristics of the formulations described in Table 17.

[0028] Figure 14 Dissolution characteristics of the formulations described in Table 18.

[0029] Figure 15A Postgastric survival rate of Saccharomyces boulardii. Survival rate of strains using the formulations described in Table 19.

[0030] Figure 15B Survival rate of a single strain (unprepared).

[0031] Figure 16 The disintegration kinetics of the formulations described in Table 19.

[0032] Figure 17 Dissolution characteristics of the formulations described in Table 20.

[0033] Figure 18 Dissolution characteristics of the formulations described in Table 21.

[0034] Figure 19 Dissolution characteristics of the formulations described in Table 22.

[0035] Figure 20 Dissolution characteristics of the formulations described in Table 23.

[0036] Figure 21 Dissolution characteristics of the formulations described in Table 24.

[0037] Figure 22 Dissolution characteristics of the formulations described in Table 25.

[0038] Figure 23 The postgastric proteolytic enzyme activity of the formulations described in Table 25.

[0039] Figure 24 Dissolution characteristics of the formulations described in Table 26. Detailed Implementation

[0040] This article describes a multifunctional oral delivery system that can be used to impart gastric protection and / or improved release to various active ingredients formulated therein. Generally, the oral delivery system uses dried particulate biopolymer components blended together and formulated with the active ingredient in a solid oral dosage form, such that the solid dosage form impregnates with gastric juices, resulting in the in-situ formation of a complex biopolymer-based aggregate. The degree of gastric protection and / or improved release to the active ingredient is determined by the structural characteristics of the in-situ formed complex aggregate, which can be controlled by the properties and ratio of the particulate / powder components applied in the oral delivery system. Furthermore, the ability of the oral delivery system described herein to be based on natural and / or naturally derived biopolymers (e.g., food biopolymers) offers a commercial advantage in terms of regulatory approval and / or meeting the growing consumer demand for such products.

[0041] This study screened a variety of natural ingredients with the aim of developing a versatile, ingredient-based oral delivery system suitable for providing gastric protection and / or improved release of various active ingredients. Empirical dissolution tests involving monitoring the release of active ingredients after successive exposure to simulated gastric and intestinal fluids demonstrated that certain mixtures of protein and polysaccharide powders can provide gastric protection for the active ingredients to which they are formulated and slow the release of the active ingredients. For example, mixtures of whey protein powder and κ-carrageenan powder (i.e., a linearly sulfated polysaccharide extracted from red edible seaweed) in tablet or capsule formulations were observed to provide enhanced protection against simulated gastric fluids for various active ingredients, as well as slow release of the active ingredients over time (Example 1). Further experiments were conducted to characterize the structure, mechanism, and reproducibility of the whey protein and κ-carrageenan powder mixtures used in the oral delivery system. More specifically, Example 2 investigated the effects of the protein / polysaccharide powder mixture ratio and native / denatured protein on the release characteristics of active ingredients in tablet formulations, and Example 3 investigated the structural characteristics of protein / polysaccharide aggregate-like complexes formed in situ after immersion in gastric fluid. The versatility of the oral delivery system for capsule formulations described herein is illustrated in Example 4.

[0042] Interestingly, while the properties and sources of the protein powders used in the oral delivery systems described herein are relatively flexible (i.e., proteins from different sources and / or suppliers can be substituted without significantly affecting gastric protection and / or improved release performance), the application of the same type of polysaccharide powder from different suppliers has produced unpredictable results. For example, κ-carrageenan powder and xanthan gum powder from different suppliers have been observed to produce opposite results in tablet erosion tests (Examples 5 and Table 6). Furthermore, without empirical dissolution testing of oral formulations, it is impossible to reliably predict in advance (e.g., based on supplier-provided product specification sheets) which supplier's polysaccharide will successfully form stable, complex aggregates in situ. These unpredictable results have led to extensive efforts to determine objective and measurable parameters that can reliably predict the polysaccharide powders suitable for the oral delivery systems for in-situ aggregate formation described herein. In particular, these efforts yielded a set of measurable / calculable parameters (i.e., dynamic cohesion index, angle of repose, compressibility index (Carl index), and / or Hausner ratio) associated with the powder flow characteristics of the polysaccharide powder, which can be used to reliably predict its suitability for the oral delivery system for in-situ agglomerate formation described herein (Table 7). Furthermore, in Example 6, a polysaccharide powder associated with poor gastric protection in the oral delivery system described herein (i.e., 100% disintegration within 12 minutes in simulated gastric fluid) underwent a conditioning step to increase its cohesiveness. The conditioning polysaccharide powder not only exhibited a more favorable parameter distribution in terms of dynamic cohesion index, angle of repose, compressibility index (Carl index), and / or Hausner ratio, but also showed a significant increase in gastric protection in the oral delivery system (i.e., only 1% disintegration in simulated gastric fluid) (Table 9). These parameters were subsequently validated for use in the oral delivery system described herein in the context of various protein powders, polysaccharide powders, active ingredients, and additives (Examples 7-22). Therefore, various aspects and implementations related to the techniques described herein are discussed below.

[0043] In some aspects, the oral delivery system described herein comprises (or substantially comprises) an active ingredient dispersed in a dry, relatively homogeneous powder mixture, which is subsequently formulated into a solid oral dosage form. The homogeneous powder mixture comprises a mixture of protein powders and polysaccharide powders, and may further comprise one or more additives (e.g., pharmaceutically acceptable excipients), depending on the specific solid oral dosage form (e.g., tablets or capsules). The protein and polysaccharide particles in the powder mixture of the oral delivery system interact, causing the oral delivery system to immerse in gastric juice or a simulated gastric juice, resulting in the in-situ formation of protein / polysaccharide complex aggregates, thereby conferring gastric protection and / or improved release of the active ingredient dispersed therein. In some embodiments, the protein and polysaccharide particles in the powder mixture of the oral delivery system may interact to form an interacting powder mixture.

[0044] As used herein, the term "oral delivery system" refers not only to a marketable product but also to a multifunctional platform for formulating various active ingredients for gastric protection and / or improved release, based on the in-situ formation of protein / polysaccharide complex aggregates as described herein. In a sense, the platform is scalable, meaning the degree of gastric protection and / or improved release can be controlled by varying the properties and / or ratios of the applied protein and polysaccharide powders. Oral delivery systems can be used to prepare specific oral dosage forms (e.g., tablets or capsules) compatible with them. For clarity, the oral delivery systems mentioned herein do not include oral dosage forms that may happen to contain polysaccharides and proteins, as well as other ingredients, where the polysaccharides and proteins are not intended to form complex aggregates in situ upon infiltration into gastric juices, or where the polysaccharides and proteins are not the primary agents responsible for gastric protection or improved release characteristics of the oral dosage form.

[0045] As used herein, in the context of the oral delivery system described herein, the phrase "mainly composed of" does not include oral dosage forms that may contain polysaccharides and proteins as well as other ingredients, but in which the polysaccharides and proteins do not form in situ complex aggregates upon infiltration into gastric juice, and in which the level of gastric protection and / or modified release (if any) imparted to the active ingredient is not determined or controlled by the strength of the formed complex aggregates.

[0046] As used herein, "aggregate" or "complex aggregate" refers to the binding, assembly, or aggregation of proteins and polysaccharides in the presence of a liquid. As reported in the scientific literature, stable aggregation typically occurs when two biopolymers with opposite charges are mixed in a liquid, usually at a pH between the pKa of the polysaccharide and the isoelectric point of the protein. Aggregation does not occur when proteins and polysaccharides are mixed in powder form in the absence of a liquid.

[0047] In some embodiments, the oral delivery system described herein can be adapted / scaled to desired levels of gastric protection and / or release rates of the active ingredient. For example, in some embodiments, changing the ratio (e.g., weight ratio) of polysaccharide powder to protein powder in a homogeneous powder mixture of the oral delivery system alters the gastric protection level and / or release rate of the active ingredient. In some embodiments, increasing the ratio (e.g., weight ratio) of polysaccharide powder to protein powder in the oral delivery system increases the level of gastric protection conferred by the oral delivery system and / or decreases the release rate of the active ingredient (e.g., in gastric juice or simulated gastric juice) – see, for example, Example 2 and Figure 1 In some embodiments, the weight ratio of the polysaccharide powder to the protein powder in the oral delivery system is 1:20 to 1:1, 1:15 to 1:1.5, 1:10 to 1:2, 1:9.5 to 1:2.5, 1:9 to 1:3, or 1:8.5 to 1:3.5, 1:8 to 1:4, 1:7.5 to 1:4.5, or 1:7 to 1:5. In some embodiments, the weight ratio is selected from any one of [1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, or 1:5] to any one of [1:4, 1:3, 1:2, or 1:1].

[0048] In some embodiments, the gastroprotection and / or improved release level of the active ingredient can be controlled by the amount of protein / polysaccharide powder mixture present in the oral delivery system. In some embodiments, the oral delivery system may include about 5% to 50%, 10% to 45%, 15% to 40%, or 20% to 35% w / w of the protein / polysaccharide powder mixture.

[0049] In some embodiments, after immersing the oral delivery system in a solution (e.g., gastric juice or simulated gastric juice) with a pH lower than the pKa of the polysaccharide, the protein and polysaccharide powder mixture forms protein / polysaccharide complex aggregates in situ. These aggregates remain intact upon subsequent incubation at a pH higher than the isoelectric point of the protein (e.g., intestinal juice or simulated intestinal juice). This is generally atypical of aggregates, as scientific literature consistently reports the formation of stable protein / polysaccharide aggregates within a pH range between the pKa of the polysaccharide and the isoelectric point of the protein material (Syrbe et al., 1998; Tolstoguzov, 1997). In some embodiments, after immersing the oral delivery system in simulated gastric juice (SGF), the protein and polysaccharide powder mixture forms protein / polysaccharide complex aggregates in situ. This simulated gastric juice consists of a 37% v / v diluted HCl solution at pH 1.0 containing 2 g / L NaCl and 0.1 g / L pepsin.

[0050] In some embodiments, the gastric protection and / or improved release level of the active ingredient after immersion in gastric juice is determined by the structural characteristics (i.e., strength) of the protein / polysaccharide complex aggregates formed in situ. In some embodiments, the release rate of the active ingredient is inversely proportional to the strength of the protein / polysaccharide complex aggregates formed in situ after immersion of the oral delivery system in gastric juice. In some embodiments, the protein / polysaccharide complex aggregates formed in situ after immersion of the oral delivery system in SGF are characterized by the presence of intramolecular β-sheets, α-helices, and / or disordered structures (e.g., measured by Fourier transform infrared (FTIR) spectroscopy; such as by the method described in Example 3).

[0051] In some embodiments, the oral delivery system described herein preferably comprises a dry, relatively homogeneous powder mixture comprising a mixture of protein powder and polysaccharide powder, wherein the polysaccharide powder has powder flow characteristics capable of forming in situ protein / polysaccharide complex aggregates upon immersion in gastric fluid of sufficient strength to provide gastric protection and / or improve release of the active ingredient to which it is formulated. In some embodiments, the polysaccharide powder is characterized by its flowability, texture, and / or cohesiveness, as measured by its angle of repose (α), dynamic cohesion index, Hausner ratio, and / or compressibility index (Carl index), according to USP. <1174> The recommended method is described below. In some embodiments, polysaccharide powders with enhanced cohesiveness (or reduced flowability) are used to form protein / polysaccharide aggregates with enhanced strength, the enhanced cohesiveness being measured by angle of repose, dynamic cohesion index, Hausner ratio, and / or compressibility index (Carl index). In some embodiments, enhanced aggregate strength may occur when the polysaccharide and protein have groups with opposite charges.

[0052] The "angle of repose" typically refers to the steepest angle between a material and a horizontal plane, measured statically or dynamically, at which the material can be stacked without collapsing (Beakawi Al-Hashemi, H. et al., 2018). The angle of repose can be determined using methods such as the "tilt box method," "fixed funnel method," "rotating drum / roller method," or GranuHeap method. TM (GranuTools TM The measurement is performed in Awan, Belgium. The maximum angle of repose is 90 degrees. Lower angles of repose (e.g., values ​​below 35 degrees) correspond to more free-flowing materials. Higher angles of repose (e.g., above 35 degrees) indicate powders with higher cohesion (non-flowability). According to USP... <1174> The suggestion is that the angle of repose can also be measured by forming a cone-shaped powder on a stable base.

[0053] In some embodiments, the polysaccharide powder used in the oral delivery system described herein has an angle of repose associated with powders having general (36-40 degrees), passable (41-45 degrees), poor (46-55 degrees), very poor (56-65 degrees), or very very poor (56-65 degrees) flowability, according to the Carr classification (Carr, RL, 1965), in USP <1174> Repeated in, such as via USP <1174> The recommended method for measurement involves, in short, allowing powder to pass through a funnel onto a fixed base with a fixed lip to retain a layer of powder on the base, thus forming a symmetrical powder cone; varying the height of the funnel to maintain a distance of approximately 2-4 cm from the top of the powder pile as it forms; and determining the angle of repose by measuring the height of the powder cone and calculating the angle of repose according to the following equation:

[0054]

[0055] In some embodiments, the polysaccharide powder used in the oral delivery system described herein has an angle of repose greater than about 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 degrees, according to USP. <1174> The method recommended in the text is used for measurement.

[0056] In some embodiments, the polysaccharide powder used in the oral delivery system described herein has a dynamic cohesion index above a threshold. "Dynamic cohesion index," as used herein, refers to a qualitative assessment of the surface slope variation of a material. The dynamic cohesion index can also be measured by instruments such as rheometers like the GranuDrum. TM (GranuTools TM The dynamic cohesive index (VCI) is measured in Awan, Belgium. A higher VCI value (e.g., between 30 and 60) indicates a material with higher cohesiveness. A lower VCI value (e.g., between 0 and 30) indicates a material with lower cohesiveness. In some embodiments, the polysaccharide powder used in the oral delivery system described herein has a VCI greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0057] In some embodiments, the polysaccharide powder used in the oral delivery system described herein has a compressibility index (Carl index) above a threshold. "Carl index" or "compressibility index," as used herein, refers to the compressibility of a powder or material, such as the USP index. <1174> As described in the text and measured using the following formula:

[0058]

[0059] Where, ρ 振实 and ρ 堆积 These refer to the tap density and bulk density of the powder, respectively. A higher compressibility index (e.g., an index exceeding 15%) indicates weaker powder flowability (higher cohesion). A lower compressibility index (e.g., an index below 15%) indicates stronger powder flowability (lower cohesion). In some embodiments, the polysaccharide powder used in the oral delivery system described herein has a compressibility index, such as USP, associated with powders having general (16-20%), passable (21-25%), poor (26-31%), very poor (32-37%), or very very poor (greater than 38%) flowability. <1174> As described herein. In some respects, the polysaccharide powder used in the oral delivery system described herein has a compressibility index greater than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38%, as measured by the methods described above.

[0060] In some embodiments, the polysaccharide powder used in the oral delivery system described herein has a Hausner ratio higher than a threshold. "Hausner ratio," as used herein, refers to the flowability of the powder, such as in USP... <1174> As described in [the text], and measured using the following formula:

[0061]

[0062] Where, ρ 振实 and ρ 堆积These refer to the tap density and bulk density of the powder, respectively. A higher Hausner ratio (e.g., above 1.18) indicates weaker powder flowability (higher cohesion). A lower Hausner ratio (e.g., below 1.18) indicates stronger powder flowability (lower cohesion). In some embodiments, the polysaccharide powder used in the oral delivery system described herein has a Hausner ratio associated with powders having general (1.19-1.25), passable (21-25%), poor (26-31%), very poor (32-37%), or very very poor (greater than 38%) flowability, such as USP. <1174> As described herein. In some embodiments, the polysaccharide powder used in the oral delivery system described herein has a Hausner ratio greater than about 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, or 1.6, for example, as measured by the methods described above.

[0063] In some embodiments, the polysaccharide powder described herein may be adjusted to have sufficient cohesiveness before being formulated into the oral delivery system described herein, such as having one or more of the following: angle of repose (α), dynamic cohesive index, Hausner ratio, and / or compressibility index (Carl index), as described herein. In some embodiments, adjustment may include any treatment that increases the cohesiveness of the polysaccharide powder and / or reduces its flowability. For example, adjustment may include one or more of dissolving, lyophilizing, drying, milling, and / or sieving the polysaccharide powder through a mesh of appropriate size to increase the powdery texture and its cohesiveness.

[0064] In some embodiments, the oral delivery system described herein can inhibit the release of the active ingredient into gastric juice and prolong, delay, sustain, control, slow, or delay the release of the active ingredient into intestinal juice. As used herein, the term "modified release" refers to the property of a solid oral dosage form to prolong, delay, slow, or control the release of the active ingredient. In some embodiments, modified release occurs after the oral delivery system is immersed in gastric juice to form a protein / polysaccharide complex aggregate.

[0065] In some embodiments, the oral delivery system described herein may be a delayed-release oral delivery system, an extended-release oral delivery system, an oral delivery system that provides enhanced gastric protection of the active ingredient after oral administration compared to administration of the unformulated active ingredient; or any combination thereof. In some embodiments, the delayed-release oral delivery system may delay the time required for 50% release of the active ingredient by at least 30, 60, 90, 120, 150, 180, 210, or 240 minutes compared to a corresponding oral delivery system lacking polysaccharide powder. In some embodiments, the extended-release oral delivery system may result in release of the active ingredient for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours. In some embodiments, the oral delivery system may provide enhanced gastric protection of the active ingredient compared to administration of the unformulated active ingredient. In some embodiments, the aforementioned improved release and / or gastric protection values ​​may refer to values ​​obtained according to the following dissolution tests: including immersing the oral delivery system in SGF for 2 hours at 37°C, followed by immersion in simulated intestinal fluid (SIF), wherein the SGF consists of a 37% v / v diluted HCl solution containing 2 g / L NaCl and 0.1 g / L pepsin, pH 1.0; and the SIF consists of a 50 mM NaH2PO4 or KH2PO4 buffer solution at pH 6.9 containing 0.5 g / L pancreatic enzyme.

[0066] In some embodiments, the protein powder used in the oral delivery system described herein may include or consist of the following: natural protein; food-grade and / or pharmaceutical-grade protein; native protein; denatured protein (e.g., heat-denatured protein); unmodified protein; chemically modified protein (e.g., succinylated protein); plant protein (e.g., cannabis, sacha inchi); animal protein; milk protein (e.g., whey protein); legume protein (e.g., pea or soy protein); fruit protein (e.g., coconut); cereal protein (e.g., rice); or any mixture thereof.

[0067] In some embodiments, the polysaccharide powder used in the oral delivery system described herein may include or consist of the following: natural polysaccharides; food-grade and / or pharmaceutical-grade polysaccharides; unmodified polysaccharides; chemically modified polysaccharides; plant polysaccharides; animal polysaccharides; polysaccharides containing negatively charged / acidic groups (anionic polysaccharides); carrageenan (e.g., κ-carrageenan); xanthan gum; alginate; pectin powder; agar, gellan gum, guar gum, carboxymethyl cellulose, sophora bean gum, mannan, glucomannan, hyaluronic acid, tamarind gum, psyllium husk gum, tara gum, acacia gum, gum arabic, gat gum, astragalus gum, ebony gum, cinnamon gum, rhamnose gum, Brunei gum, *Pseudomonas macrocaryophyllus* gum, gel polysaccharide, *Pseudomonas breviculatus* polysaccharide, fucoidan, or any mixture thereof.

[0068] As used herein, “active ingredient” means any molecule, substance, or microorganism having therapeutic or biological activity. In some embodiments, the active ingredient described herein may include or consist of: dietary supplements, pharmaceuticals (e.g., caffeine), probiotics (e.g., probiotic bacteria or yeasts such as Saccharomyces boulardii or Pediococcus acidilactici), prebiotics, vitamins (e.g., vitamin B2 / riboflavin or B6), amino acids (e.g., 5-hydroxytryptophan [5-HTP]), food or plant extracts (e.g., peppermint extract, rice extract, rice husk extract, or curcumin), or herbal supplements (e.g., goldenrod extract and ginger). In some embodiments, the active ingredient may be in powder or granule form.

[0069] In some embodiments, the oral delivery system described herein may further include one or more nutritionally or pharmaceutically acceptable excipients and / or additives (e.g., fillers, binders, lubricants, flow agents). Such excipients and / or additives vary depending on the type of oral dosage form (e.g., tablets or capsules) formulated. In some embodiments, excipients and / or additives may include microcrystalline cellulose, magnesium stearate, stearic acid, calcium hydrogen phosphate, calcium carbonate, dextrose, and / or silica.

[0070] In some embodiments, the oral delivery system described herein may include a homogeneous blend of protein powder, polysaccharide powder, and active ingredient, which is compressed into tablets. In some embodiments, the homogeneous blend includes one or more pharmaceutically acceptable excipients or additives (e.g., as described herein), which may be fillers, binders, lubricants, and / or flow agents.

[0071] In some embodiments, the oral delivery system described herein may include a homogeneous blend of protein powder, polysaccharide powder, and active ingredient contained in a capsule (e.g., gel- or cellulose-based). In some embodiments, the capsule may be a macrocapsule. In some embodiments, the capsule may be a microcapsule.

[0072] In some embodiments, the oral delivery system described herein preferably does not include enteric coating.

[0073] In some aspects, this document describes a method for preparing a solid oral dosage form, the method comprising dispersing an active ingredient in a dry, homogeneous mixture comprising protein powder and polysaccharide powder, and formulating the resulting mixture into a solid oral dosage form. In some embodiments, the polysaccharide powder has powder flow characteristics capable of interacting with the protein powder, such that the solid oral dosage form, upon immersion in gastric juice, results in the in-situ formation of protein / polysaccharide complex aggregates, thereby imparting gastric protection and / or modified release of the active ingredient.

[0074] In some embodiments, the methods described herein apply polysaccharide powders having one or more powder flow characteristics as defined herein (e.g., related to dynamic cohesion index, angle of repose, compressibility index (Carl index), and / or Hausner ratio).

[0075] In some embodiments, the methods described herein are used to prepare the oral delivery system described herein.

[0076] In some respects, this document describes an oral delivery system as described herein, or an oral dosage form produced by methods as described herein, for therapeutic use. In some embodiments, treatment is the improvement of a disease or condition by oral administration of an active ingredient formulated in an oral delivery system or oral dosage form as described herein.

[0077] In some respects, this document describes a method for in situ generation of protein / polysaccharide complex aggregates that imparts gastric protection and / or modified release to the active ingredient dispersed therein. The method includes providing the active ingredient formulated in an oral delivery system according to the present invention or in an oral dosage form produced according to the method described herein, and orally administering the oral delivery system or oral dosage form to a subject, wherein the protein and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ after the oral delivery system is immersed in the subject's gastric juice.

[0078] In some respects, this document describes methods for treating diseases or conditions that improve the disease or condition by administering to a subject an active ingredient that benefits from gastric protection and / or modified release. The method includes providing the active ingredient formulated in an oral delivery system according to the present invention or in an oral dosage form manufactured according to the methods described herein, and orally administering the oral delivery system or oral dosage form to a subject, wherein, after immersing the oral delivery system in the gastric juice of the subject, a protein / polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ.

[0079] project

[0080] 1. An oral delivery system comprising (or substantially consisting of): a dried homogeneous mixture comprising a mixture of protein powder and polysaccharide powder and an active ingredient dispersed therein, wherein, upon immersion of the oral delivery system in gastric juice, the protein and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ, thereby imparting gastric protection to the active ingredient and / or improved release of the active ingredient, wherein changing the ratio of protein powder to polysaccharide powder in the oral delivery system alters the level of gastric protection of the active ingredient and / or the release rate of the active ingredient.

[0081] 2. The oral delivery system according to Item 1, wherein the polysaccharide powder has or is adjusted to have one or more of the following powder flow characteristics: (a) an angle of repose (α) greater than about 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59. (a) 60, 61, 62, 63, 64 or 65 degrees; (b) Dynamic cohesion index greater than approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50; (c) Compressibility The index (Carl index) is greater than approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38%; (d) the Hausner ratio is greater than approximately 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, or 1.3. 1, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59 or 1.6; or any combination of (e)(a) to (d).

[0082] 3. The oral delivery system according to Item 2, wherein the polysaccharide powder has or is adjusted to have all the powder flow characteristics defined in (a), (b), (c) and (d).

[0083] 4. An oral delivery system according to any one of items 1 to 3, wherein the protein and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ after the oral delivery system is immersed in a solution with a pH lower than the pKa of the polysaccharide.

[0084] 5. An oral delivery system according to any one of items 1 to 4, wherein: (a) the ratio of polysaccharide powder to protein powder in the oral delivery system is increased to increase the level of gastric protection for the active ingredient and / or decrease the release rate of the active ingredient; (b) the weight ratio of polysaccharide powder to protein powder in the oral delivery system is 1:20 to 1:1, 1:15 to 1:1.5, 1:10 to 1:2, 1:9.5 to 1:2.5, 1:9 to 1:3 or 1:8.5 to 1:3.5, 1:8 to 1:4, 1:7.5 to 1:4.5 or 1:7 to 1:5; (c) the oral delivery system comprises about 5% to 50%, 10% to 45%, 15% to 40% or 20% to 35% w / w of the protein and polysaccharide mixture; or (d) any combination thereof.

[0085] 6. An oral delivery system according to any one of items 1 to 5, wherein: (a) after immersing the oral delivery system in simulated gastric juice (SGF), the protein and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ, the simulated gastric juice consisting of a diluted HCl solution of pH 1.0 at 37% v / v containing 2 g / L NaCl and 0.1 g / L pepsin; (b) the release rate of the active ingredient is inversely proportional to the intensity of the protein / polysaccharide complex aggregate formed in situ after immersing the oral delivery system in the gastric juice; (c) the protein / polysaccharide complex aggregate formed in situ after immersing the oral delivery system in the SGF is characterized by the presence of intramolecular β-sheets, α-helices, and / or disordered structures (e.g., measured by Fourier transform infrared (FTIR) spectroscopy); or (d) any combination thereof.

[0086] 7. An oral delivery system according to any one of items 1 to 6, wherein the oral delivery system is: (i) a delayed-release oral delivery system; (ii) a prolonged-release oral delivery system; (iii) an oral delivery system that provides enhanced gastric protection for said active ingredient after oral administration compared with administration of unformed active ingredient; or (iv) any combination thereof.

[0087] 8. The oral delivery system according to item 7, wherein: (i) compared to a corresponding oral delivery system lacking the polysaccharide powder, the delayed-release oral delivery system delays the time required for the release of 50% of the active ingredient by at least 30, 60, 90, 120, 150, 180, 210, or 240 minutes; (ii) the extended-release oral delivery system results in the release of the active ingredient for a period of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours; and / or (ii) compared to... Compared to administering the unformulated active ingredient, the oral delivery system provides enhanced gastric protection for the active ingredient; this is based on dissolution testing, which includes immersing the oral delivery system in SGF at 37°C for 2 hours, followed by immersion in simulated intestinal fluid (SIF), wherein the SGF consists of a 37% v / v diluted HCl solution containing 2 g / L NaCl and 0.1 g / L pepsin, pH 1.0; and the SIF consists of a 50 mM NaH2PO4 or KH2PO4 buffer solution at pH 6.9 containing 0.5 g / L pancreatin.

[0088] 9. An oral delivery system according to any one of items 1 to 8, wherein the protein powder comprises or is composed of the following: natural proteins; food-grade and / or pharmaceutical-grade proteins; native proteins; denatured proteins (e.g., heat-denatured proteins); unmodified proteins; chemically modified proteins (e.g., succinylated proteins); plant proteins (e.g., cannabis, inchi); animal proteins; milk proteins (e.g., whey protein); legume proteins (e.g., pea or soy protein); fruit proteins (e.g., coconut); cereal proteins (e.g., rice); or any mixture thereof.

[0089] 10. An oral delivery system according to any one of items 1 to 9, wherein the polysaccharide powder comprises or is composed of the following: natural polysaccharides; food-grade and / or pharmaceutical-grade polysaccharides; unmodified polysaccharides; chemically modified polysaccharides; plant polysaccharides; animal polysaccharides; polysaccharides containing negatively charged / acidic groups (anionic polysaccharides); carrageenan (e.g., κ-carrageenan); xanthan gum; alginate; pectin powder; agar, gellan gum, guar gum, carboxymethyl cellulose, locust bean gum, mannan, glucomannan, hyaluronic acid, tamarind gum, psyllium husk gum, tara gum, acacia gum, gum arabic, gat gum, astragalus gum, ark tussock gum, cinnamon gum, rhamnose gum, Brunei gum, large stem mycorrhizal gum, gel polysaccharide, short-stem mycorrhizal, fucoidan, or any mixture thereof.

[0090] 11. The oral delivery system according to any one of items 1 to 10, wherein the active ingredient is a dietary supplement, a medicine, a probiotic, a vitamin, an amino acid, a food extract, or a herbal supplement.

[0091] 12. The oral delivery system according to any one of items 1 to 11, further comprising one or more nutritionally or pharmaceutically acceptable excipients and / or additives (e.g., fillers, binders, lubricants and / or flow agents).

[0092] 13. The oral delivery system according to item 12, wherein the additive is or includes the following: microcrystalline cellulose, magnesium stearate and / or silicon dioxide.

[0093] 14. The oral delivery system according to any one of items 1 to 13, wherein the oral delivery system is a tablet or capsule.

[0094] 15. The oral delivery system according to any one of items 1 to 14, wherein the oral delivery system does not include enteric coating.

[0095] 16. A method for preparing a solid oral dosage form, the method comprising dispersing an active ingredient in a dry, homogeneous mixture comprising a protein powder and a polysaccharide powder, and formulating the resulting mixture into a solid oral dosage form, wherein the polysaccharide powder has powder flow characteristics and is capable of interacting with the protein powder such that immersing the solid oral dosage form in gastric juice results in the in-situ formation of a protein / polysaccharide complex aggregate, thereby imparting gastric protection to the active ingredient and / or improved release of the active ingredient.

[0096] 17. The method according to item 16, wherein the polysaccharide powder has one or more of the powder flow characteristics defined according to item 2.

[0097] 18. The method according to item 16 or 17, wherein the solid oral dosage form is an oral delivery system defined according to any one of items 1 to 15.

[0098] 19. The use of an oral delivery system as defined in any one of items 1 to 15, or an oral dosage form produced by any one of items 16 to 18, for therapeutic purposes.

[0099] 20. A method for in situ generation of a protein / polysaccharide complex aggregate, the protein / polysaccharide complex aggregate imparting gastric protection to an active ingredient dispersed therein and / or improved release of the active ingredient, the method comprising providing the active ingredient in an oral delivery system defined according to any one of items 1 to 15 or in an oral dosage form produced by the method according to any one of items 16 to 18, and orally administering the oral delivery system or the oral dosage form to a subject, wherein the protein and polysaccharide powder mixture forms the protein / polysaccharide complex aggregate in situ after the oral delivery system is immersed in the gastric juice of the subject.

[0100] 21. A method for treating a disease or condition, the method being improved by administering to a subject an active ingredient that benefits from gastric protection and / or modified release, the method comprising providing the active ingredient in an oral delivery system defined according to any one of items 1 to 15 or in an oral dosage form produced by the method according to any one of items 16 to 18, and orally administering the oral delivery system or the oral dosage form to a subject, wherein, after immersing the oral delivery system in the gastric juice of the subject, the protein and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ.

[0101] Example

[0102] Example 1:

[0103] Screening natural ingredients for protein-based oral delivery systems

[0104] To develop a relatively simple modified release oral delivery system based on natural ingredients, extensive screening was conducted using a variety of natural ingredients for manufacturing solid oral dosage forms. Specifically, food-grade natural ingredients from different sources and suppliers, alone or in various combinations, were formulated with different active ingredients (all in dry form) into tablet or capsule oral formulations. Dissolution tests were then performed on the oral formulations, including sequential exposure to simulated gastric and intestinal fluids, followed by monitoring of the release of the active ingredient over time. Natural ingredients that could be used in protein-based oral formulations were particularly emphasized in the screening to achieve a degree of gastric protection and / or slow release of the active ingredient.

[0105] Interestingly, the results of extensive screening efforts indicate that certain mixtures of protein and polysaccharide powders formulated with various active ingredients can provide gastric protection and slow release of the active ingredients in oral formulations. For example, mixtures of whey protein powder and κ-carrageenan powder (i.e., linearly sulfated polysaccharides extracted from red edible seaweed) in tablet or capsule formulations have been observed to provide enhanced protection against simulated gastric juices for various active ingredients, as well as slow release of the active ingredients over time. Further experiments characterizing the structure, mechanism, and reproducibility of whey protein and κ-carrageenan powder mixtures for gastric protection and / or modified release oral delivery systems were conducted as described in the examples below.

[0106] Example 2:

[0107] The effect of protein / polysaccharide powder mixture ratio and native / denatured protein on the release characteristics of active ingredients in tablet formulations.

[0108] 2.1 Tablet Manufacturing

[0109] All ingredients in this embodiment, except whey protein, were from the same supplier, hereinafter referred to as Supplier A. Riboflavin (vitamin B2) was selected as the active ingredient. Native whey protein (Agropur Ingredients, Le Sueur, MN, USA) was heat-denatured at 85°C for 30 min. Tablets were prepared by direct compression using a TDP-6 single-punch press (Yangzhou Nuoya Machinery Co., Ltd., Jiangju, China). The powder was weighed before tableting and then mixed together in a mortar. The tablet diameter and thickness were 11.2 mm and 6.5 mm, respectively. 24 h after manufacturing, tablet hardness was tested using a YD-1 tablet hardness tester (Minsheng Pharmaceutical Machinery Ltd., Shanghai, China) according to the United States Pharmacopeia (USP). <1217> Measure the tablet hardness. The hardness is between 7 and 9 kPa. The formulations tested in this example are described in Table 1.

[0110] Table 1: Tablet formulations tested in Example 2

[0111]

[0112]

[0113] 2.2 Tablet dissolution test

[0114] The in vitro properties of the tablets were tested using an SR6 dissolution tester at USPII (Hanson Research Corp., Chatsworth, CA). This was conducted in accordance with USP... <2040> The guidelines described in [the document] (disintegration and dissolution of dietary supplements).

[0115] During the experiment, the paddle speed was set to 100 rpm, and the temperature was maintained at 37°C. Before measurement, the sample was filtered and background absorbance was subtracted. After release, the concentration of the active ingredient (AI) dissolved in the release medium was measured over time. Absorbance at 440 nm was tracked using a UV-Vis spectrophotometer.

[0116] A typical dissolution test consists of the following steps: immersing three tablets in simulated gastric fluid (SGF) for 2 hours, followed by immersion in simulated intestinal fluid (SIF) until complete dissolution. No settling device is used.

[0117] SGF consisted of a diluted HCl (37%) solution containing 2 g / L NaCl and 0.1 g / L pepsin, pH 1.0. SIF consisted of a 50 mM buffer solution containing 0.5 g / L trypsin in NaH₂PO₄, pH 6.9. Experiments were repeated at least twice.

[0118] 2.3 In vitro riboflavin release in tablet dissolution test

[0119] Figure 1 The in vitro release profiles obtained from formulations 2-1 to 2-7 are shown (see Table 1). Rapid release of riboflavin from 20 tablets (formulation 2-1) without polysaccharides: complete release of riboflavin and tablet dissolution occurred after 45 min.

[0120] Tablets containing both native whey protein and κ-carrageenan showed slower riboflavin release profiles. After 2 hours, the release percentages of formulations 2-2, 2-3, and 2-4 were 68%, 41%, and 24%, respectively. Complete release occurred after 225 min, 330 min, and 330 min for formulations 2-2, 2-3, and 2-4, respectively. These results indicate that the presence of the polysaccharide (κ-carrageenan) reduces the riboflavin release rate. Furthermore, these results suggest that a higher polysaccharide-to-protein ratio corresponds to a lower release rate of the active ingredient.

[0121] Replacing the native whey protein in formulations 2-2, 2-3, and 2-4 with the corresponding amount of denatured whey protein (i.e., formulations 2-5, 2-6, and 2-7) resulted in an even slower release rate of the active ingredient. In fact, after 8 hours of testing, the riboflavin release from formulations 2-5 to 2-7 was between 65% and 70%. Figure 1It is noteworthy that the use of denatured proteins results in a particularly slow release of riboflavin during SGF exposure (0 to 120 min). Figure 1 ).

[0122] Example 3:

[0123] Protein / polysaccharide powder mixtures form aggregates in situ.

[0124] To investigate the structural and physicochemical properties of the tablet formulation of Example 2 after exposure to SGF and SIF, at least two types of tests were performed: intensity tests with and without dispersants; and Fourier transform infrared (FTIR) spectroscopy.

[0125] 3.1 Strength Measurement with or without Discriminant

[0126] The tablets prepared as described in Example 2.1 were immersed in 50 mL of SGF for 2 hours, or in 50 mL of SGF for 2 hours, followed by immersion in SIF for 2 hours, as described in Example 2.2. Although all formulations 2-2 to 2-7 formed seemingly aggregate-like complexes during the incubation process, only formulations 2-5, 2-6, and 2-7 were strong enough for physical manipulation and were therefore selected for further study.

[0127] Using a 25kg load sensor (Stable Micro Systems, Scarsdale, NY, USA) and a cylindrical steel probe. The TA-XT2 texture analyzer was used to measure the strength of aggregated complexes of formulations 2-5, 2-6, and 2-7 after immersion. The resistance of the samples to probe compression was recorded. The pre-test speed was set to 2.0 mm / s, and the test speed was set to 0.2 mm / s. The post-test speed was set to 1.0 mm / s. The acquisition rate was 10 points per second. An example of stress-strain curves is shown below. Figure 2 As shown. Furthermore, to evaluate the contributions of hydrophobic interactions and hydrogen-structure bonding to the condensate-like structure, experiments were repeated using SGF and SIF containing 2M ethanol or 2M urea, respectively. For each sample, the cohesive work was calculated as the area under the stress-strain curve between 0 and 2.5 mm. Cohesive work is expressed in mJ. Typical results are shown in Table 2. All experiments were repeated at least three times.

[0128] Table 2: SGF or S in the presence and absence of a dispersant GF / SIF The cohesive work (in terms of) of formulations 2-5, 2-6 and 2-7 in the formula 10 -4 mJ (measured)

[0129]

[0130] After 2 hours of immersion in SGF, the higher the polysaccharide-to-protein ratio used in the formulation, the higher the observed cohesive work. This result indicates that a higher ratio of polysaccharide (e.g., κ-carrageenan) leads to stronger aggregates. It also suggests that the strength of the complex after immersion is related to polysaccharide-protein interactions, and further, an increased polysaccharide-to-protein ratio in the formulation may favor protein / polysaccharide interactions, but at the expense of protein-protein interactions. The cohesive work value tends to decrease in the presence of dispersants. Therefore, both urea and ethanol lead to a decrease in the strength of aggregate-like complexes, indicating that both hydrogen bonding and hydrophobic forces affect the stability of the complex. Furthermore, the higher the polysaccharide-to-protein ratio, the more pronounced the effect of the dispersant. This result confirms that the strength of the complex strongly depends on protein / polysaccharide interactions, indicating that not only does aggregate formation occur, but the strength of the formed aggregates is inversely proportional to the release rate of the active ingredient. Unbound by theory, aggregates can significantly slow down the release of the active ingredient by forming a tighter and stronger complex.

[0131] After immersion in SGF for 2 hours, followed by immersion in SIF for 2 hours, increasing the polysaccharide-to-protein ratio resulted in a slight increase in cohesive work (formulations 2-5 to 2-6), followed by a decrease in complex strength of approximately 50% in formulations 2-7. However, despite these minor differences, the effect of the dispersant remained particularly significant, indicating that protein / polysaccharide interactions determine the strength of aggregate-like complexes, even after subsequent incubation in SIF.

[0132] Interestingly, although the dissociation constant of the sulfate group of κ-carrageenan is approximately 3 (pKa ~ 2.8) and the isoelectric point of whey protein is approximately 5 (pI ~ 5.2), the aggregate complexes remained intact after incubation at pH 6.9 (SIF). Therefore, the formation of aggregate-like complexes in formulations 2-5 to 2-6 after immersion in SGF (and SGF / SIF) is somewhat unexpected, as the scientific literature consistently reports that stable protein / polysaccharide aggregates form between the pKa of the polysaccharide and the isoelectric point (pI) of the protein material (Syrbe et al., 1998; Tolstoguzov, 1997). Thus, these results highlight the influence of the protein / polysaccharide ratio on the strength of aggregate-like complexes in both SGF (pH 1.0) and SIF (pH 6.9).

[0133] 3.2 FTIR analysis of the amide I' region

[0134] The formulation was incubated for 2 h in 10 mL of D₂O adjusted to pD 1.5 (pH 1.1) with 0.5 M DCl; or incubated for 2 h in the same solution, followed by incubation in 10 mL of D₂O adjusted to 7.3 (pH 6.9) with 0.5 M NaOD. The tablet surface was then gently pressed and analyzed using horizontally attenuated total reflectance (ATR) crystals (ZnSe). Infrared spectra were recorded using a Magna 560 Nicolet spectrometer (Madison, WI, USA) equipped with a mercury-cadmium-telluride detector. The spectrometer was continuously purged with dry air. Each spectrum is the result of an average of 128 scans and apodization was performed using the Happ-Genzel function. Omnic spectra were used to investigate the amide I' region. TM The software performs subtraction and Fourier deconvolution. Bandwidth narrowing is achieved, with a full width of 18cm. -1 At halfway point and with a resolution enhancement factor of 2cm -1 All spectra were analyzed at least twice.

[0135] Figure 3 The results of TIR analysis are shown, illustrating the amide I' regions of aggregates on the surfaces of tablet formulations 2-5, 2-6, and 2-7, as described in Example 3.1. Interestingly, no particular differences were observed between the spectra obtained at acidic (SGF) or more neutral pH (SGF followed by SIF). In fact, no differences were observed in band position or intensity when formulations 2-5, 2-6, and 2-7 were immersed in SGF, followed by or without immersion in SIF. These results suggest that aggregate-like complexes initially form upon contact with SGF during the gastric phase, and these complexes remain relatively structurally unchanged upon subsequent exposure to SIF.

[0136] More in detail, Figure 3 This indicates that all spectra include those located at 1695 cm⁻¹. 1 1681cm -1 1671 -1 1669cm -1 1662-1660cm -1 1648cm -1 1632cm -1 1622-1617cm -1 and 1603cm -1 The eight components are located at 1632, 1662-1660, 1671-1669, and 1691 cm. -1 The components at this location originate from intramolecular β-sheets and disordered structure. Located at 1648 cm⁻¹ -1 The band at this location originates from α-helices and a disordered structure. 1603cm-1 The small shoulder at this location originates from the vibration of the amino acid side chain. It is located at approximately 1617-1622 cm. -1 The amide I' maximum value at this point is characterized by the presence of intermolecular β-sheet hydrogen bonds. Such a band is characteristic of protein-protein interactions. The frequency of this band appears to gradually decrease from 1617 cm⁻¹. -1 (Formulation 2-5) moved to 1622cm -1 (Formulations 2-7) (Gilbert et al., 2005). This may be due to the weakening of protein-protein interactions as the polysaccharide concentration increases in the tablet. Unbound by theory, this phenomenon may be related to the insertion of polysaccharide (κ-carrageenan) molecules between protein chains. This result is consistent with our other observations that an increase in the polysaccharide ratio in the tablet favors protein-polysaccharide interactions at the expense of protein-protein interactions. Finally, located at 1681 cm⁻¹ -1 The less obvious components at that point would be consistent with the presence of antiparallel intermolecular β-sheets.

[0137] In general, these results provide compelling evidence that in situ protein / polysaccharide complex aggregates form upon immersion of formulations in SGF (pH 1.0) and that these aggregates can adequately withstand subsequent exposure to SIF (pH 6.9) to achieve modified / slow release of the active ingredient. Furthermore, it was observed that aggregate strength and the release rate of the active ingredient can be controlled by simply altering the ratio of the protein / polysaccharide mixture during formation. This suggests that the release rate and / or duration of the active ingredient can be tailored by varying the biopolymer ratio in oral formulations, thus providing a basis for versatile aggregate-based oral delivery systems that are easily adaptable to desired release profiles.

[0138] Example 4:

[0139] Protein / polysaccharide powder mixtures that form aggregates are suitable for capsule formulations.

[0140] Capsules are one of the most commonly used solid oral dosage forms for delivering active ingredients and offer several advantages over tablets for certain formulations. In this example, a protein / polysaccharide powder mixture that forms aggregates was added to a capsule formulation to evaluate its suitability for such formulations.

[0141] 4.1 Capsule Preparation

[0142] Using cellulose-based capsules (HPMC, K-Caps) TMEmpty capsules were manually filled with a mixture of protein / polysaccharide powders, consisting of pea protein powder (The Scoular Company, Nebraska, USA) mixed with carrageenan (supplier A) and xanthan gum (supplier D) powders, microcrystalline cellulose (MCC), and the active ingredient (caffeine or probiotic Pediococcus acidilactici). The protein:polysaccharide mixture had a weight ratio of 85:15. The capsules were then manually sealed with caps. The in vitro release properties of the capsules were subsequently tested.

[0143] 4.2 Capsule in vitro release performance

[0144] The in vitro release performance of the capsules was tested using an SR6 dissolution tester (Hanson Research Corp., Chatsworth, CA). For each formulation, dissolution was performed according to USP. <724> Dissolution specification tests were performed on six capsules for the delayed-release formulation. In brief, a typical dissolution test involved immersing the capsules in simulated gastric fluid (SGF) for 2 hours, followed by immersion in simulated intestinal fluid (SIF) until complete dissolution. The paddle speed was set to 65 rpm, and the temperature was maintained at 37°C. The container volume was 1000 mL.

[0145] SGF consisted of a diluted HCl (37%) solution containing 2 g / L NaCl and 0.1 g / L pepsin, pH 1.0. SIF consisted of a buffer solution (50 mM) of NaH₂PO₄ containing 0.5 g / L trypsin, pH 6.9. Experiments were repeated at least twice. During each experiment, a stainless steel settler with six screws was used to ensure capsule immersion.

[0146] For probiotics, *Pediococcus lactis* viability was measured after immersion in SGF for 1 h and 2 h. The capsules were then transferred and completely dissolved in 150 mL of sterile SGF. Subsequently, dilutions were performed, and the strains were cultured on MRS agar at 37°C under aerobic conditions for 48 h. Microbial counting was then conducted.

[0147] For caffeine, absorbance at 271 nm was measured after release using a UV160AUV visible spectrophotometer (Shimadzu, Kyoto, Japan). The sample was filtered prior to analysis.

[0148] 4.3 Results

[0149] Probiotic survival

[0150] The tested formulations are presented in Table 3, expressed as a percentage by weight (%). Three different formulations were prepared, comprising 0%, 35%, and 50% w / w of a protein / polysaccharide powder mixture, respectively. The final weight of the capsules was 500 ± 15 mg.

[0151] Table 3: Tested Pediococcus lactis capsule formulations

[0152]

[0153] * Pea protein powder: carrageenan / xanthan gum powder (weight ratio 85:15).

[0154] The results of probiotic survival showed Figure 4 The capsules opened approximately 12 minutes after being placed in SGF. Capsules containing only microcrystalline cellulose (MCC) (formulation 4-1) showed rapid dissolution and no detectable viability of *Pediococcus lactis* after 1 hour in SGF. Conversely, formulations 4-2 and 4-3 showed significant viability after the gastric step. After 1 hour, formulations 4-2 and 4-3 produced 7.4 ± 1.8 × 10⁻⁶ cells / mL, respectively. 9 and 6.2±2.0×10 9 Live CFU. After 2 hours, for formulation 4-2, the CFU level was 7.7 ± 3.1 × 10⁻⁶. 4 For formulation 4-3, the CFU was 1.2 ± 0.1 × 10⁻⁶. 9 Notably, while the control formulation 4-1 (a protein-polysaccharide mixture lacking aggregate formation) showed a 10.5 log decrease in viability, formulations 4-2 and 4-3 showed only a 1 log decrease in viability after 1 hour in SGF.

[0155] The observed gastric protection can be explained by the formation of aggregates from the protein / polysaccharide mixture as the capsule begins to open. Indeed, as simulated gastric juices seep into the capsule and the capsule begins to dissolve, the protein / polysaccharide mixture begins to form aggregates in situ (as is characteristic of the infiltrated tablet in Example 3), resulting in the formation of complex aggregates suited to the capsule shape (see [link to relevant documentation]). Figure 5 ).

[0156] Therefore, these results demonstrate the suitability of protein / polysaccharide mixtures that form aggregates for gastric protection of active ingredients and for improved release of active ingredients.

[0157] caffeine release

[0158] The same experiments as with *Pediococcus lactis* were performed, using caffeine instead of the active ingredient. Two formulations were tested: one containing a protein / polysaccharide mixture, and the other containing only a filler (MCC). The tested capsule formulations are described in Table 4. Each capsule weighed approximately 500 mg.

[0159] Table 4: Caffeine capsule formulations tested (in % w / w)

[0160]

[0161] * Pea protein powder: carrageenan / xanthan gum powder (weight ratio 85:15).

[0162] exist Figure 6 The release curves obtained from each formulation are presented. For both formulations, caffeine began to be released after 5 to 10 minutes in the experiment. This time period corresponds to the time required for the capsule to begin dissolving and exposing its contents to the surrounding fluid.

[0163] In formulations containing only MCC as a filler (formulation 4-4), rapid release of caffeine was observed, with 97% of the caffeine released after 15 minutes in SGF.

[0164] In formulations containing a 35% w / w protein / polysaccharide mixture (4-5), caffeine release was prolonged by several hours, with 100% release observed after approximately 6-7 hours. This result is consistent with the results for probiotics and indicates that the protein / polysaccharide powder mixture forms a complex that protects the active ingredient from acidic conditions and is able to extend the release time of the active ingredient from minutes to hours.

[0165] 4.4 Discussion

[0166] The results in this embodiment demonstrate that, in addition to tablet formulations, protein / polysaccharide powder mixtures that form aggregates in situ are also suitable for capsule formulations. In fact, their use in capsules resulted in improved release profiles, with dissolution of the active ingredient prolonged by several hours, and also provided protection of the active ingredient under simulated gastric conditions.

[0167] The dissolution of gelatin-based or cellulose-based capsules is not an instantaneous and complete process. Unbound by theory, when capsule dissolution begins, surrounding fluids may seep into the capsule, leading to the simultaneous dissolution of the active ingredient and the formation of protein / polysaccharide complex aggregates. This process continues until the capsule is completely dissolved, resulting in a structure suitable for the capsule shape (e.g., see...). Figure 5 ).exist Figure 7 The diagram schematically illustrates the process of capsule dissolution and aggregate formation of complexes.

[0168] Capsules are typically manufactured using standard techniques currently available, typically employing gelatin or cellulose. Once filled with a given formulation containing the active ingredient, additional processing steps and additives are required, such as applying a coating to the capsule surface to obtain gastric protection and / or a modified release profile. This article demonstrates that, notably, protein / polysaccharide powder mixtures that form aggregates in situ can provide gastric protection and modified release of the active ingredient without additional processing steps (such as without adding additional coatings to the capsules, e.g., enteric coatings).

[0169] Example 5:

[0170] Parameters that determine the performance of polysaccharide powders in oral delivery systems based on in-situ formation of protein / polysaccharide aggregates

[0171] Examples 1-4 demonstrate the suitability of protein / polysaccharide powder mixtures for oral delivery systems to form aggregates. More specifically, Examples 2 and 3 demonstrate aggregate formation associated with improved release of the active ingredient in both native and denatured whey proteins, while Example 4 demonstrates results comparable to pea protein powder, indicating that suitable aggregate formation can be achieved using protein powders from different sources. In each of Examples 2-4, suitable aggregate formation was demonstrated using the polysaccharide κ-carrageenan from the same supplier (Supplier A). However, extensive empirical dissolution tests using different types of polysaccharides (i.e., different from κ-carrageenan) and even the same type of polysaccharides from different suppliers (e.g., κ-carrageenan obtained from different suppliers) produced unpredictable results in terms of aggregate formation and gastric protection / improved release. (This unpredictable result was not observed when applying protein powders from different suppliers, all of which had a more uniform powder texture.) For example, κ-carrageenan powder obtained from Supplier A was observed to form complex aggregates in situ to provide gastric protection, but κ-carrageenan from Supplier B could not do so. Furthermore, without empirical dissolution testing of oral formulations, it is impossible to reliably predict in advance (e.g., based on supplier-provided product specification sheets) which polysaccharide from which supplier will successfully form aggregates in situ. These unpredictable results led to extensive efforts to determine objective and measurable parameters that can reliably predict the function of the polysaccharide powders in the oral delivery systems for in-situ aggregate formation described in Examples 1-4. These efforts yielded a set of measurable / calculable polysaccharide powder parameters (i.e., dynamic cohesion index, angle of repose, Karl index / compressibility, and Hausner ratio) that can be used to reliably predict their suitability for the oral delivery systems for in-situ aggregate formation described herein. The results shown below in this example demonstrate the foregoing.

[0172] 5.1 Tablet Manufacturing

[0173] Tablets were prepared by direct compression using a TDP-6 single-punch press (Yangzhou Nuoya Machinery Co., Ltd., Jiangju, China). The powder was weighed before compression and then mixed together in a mortar. The tablet diameter and thickness were 11.2 mm and 6.5 mm, respectively. 24 hours after manufacturing, tablet hardness was measured using a YD-1 tablet hardness tester (Minsheng Pharmaceutical Machinery Ltd., Shanghai, China) according to USP standards. <1217> Measure the tablet hardness. The hardness is between 7 and 9 kPa.

[0174] In this embodiment, heat-denatured pea protein was selected as the protein powder, and multiple polysaccharides from different suppliers were formulated in parallel. The general composition of the tablet formulation is shown in Tables 5 and 6.

[0175] Table 5: Tablet formulations tested in this example

[0176]

[0177]

[0178] 5.2 Tablet Erosion

[0179] The erosion of the tablets was tracked using a disintegration analyzer (Tianjin Guoming Medicinal Equipment Co., Tianjin, China). This was done in accordance with USP specifications. <2040> Tests were conducted. In a typical experiment, one tablet was placed in each of three test tubes in a basket. No settling tank was used. The apparatus was then run for 1 hour using SGF at 37°C as the immersion solution. The experiment was performed in at least triplicate. Disintegration was determined by gravimetric analysis. After 1 hour, the erosion rate of the tablet was measured and expressed as the percentage of disintegrated tablet (D%). If the tablet completely disintegrated before the end of the experiment, its disintegration time (DT) was recorded. Table 6 illustrates the D% and DT values ​​for different test tablets.

[0180] 5.3 Characteristics of Polysaccharide Powder

[0181] Polysaccharide powders were characterized by measuring their angle of repose, dynamic cohesion index, bulk density and tap density, compressibility index (Carl index), and Hausner ratio. Powder properties are in accordance with USP. <1174> The standard is defined. The dynamic cohesion index is assessed using the correlation between the α value (angle of repose) and the cohesion index (Boschini et al., 2015).

[0182] These properties are indicators of powder flowability, texture, and cohesion. The angle of repose (α) is closely related to the powder cohesion index (Boschini et al., 2015), therefore, the α value is used to determine the corresponding cohesion index value.

[0183] 5.4 Results

[0184] Pediococcus lactis was selected as the active ingredient. All tablets have the same formulation (see Table 5), differing only in the type or source of the polysaccharides (see Table 6).

[0185] Table 6: Tablet Disintegration D% and DT

[0186]

[0187] The results in Table 6 show that formulations 5-2, 5-3, 5-5, 5-9, 5-10, 5-11, 5-12, 5-13, and 5-14 (in bold) exhibited significant resistance to SGF after 1 hour of exposure, indicating their suitability for gastric protective / modified release applications. In contrast, formulations 5-1, 5-6, 5-7, and 5-8 provided almost no gastric resistance after 1 hour of SGF exposure, ruling out their use in gastric protective / modified release oral delivery systems. Formulation 5-4 exhibited moderate gastric resistance to SGF.

[0188] Surprisingly, formulations 5-1 and 5-2 exhibited completely opposite erosion results (100% vs. 5%), despite being composed of all the same ingredients. In fact, the only difference between formulations 5-1 and 5-2 was the source of the polysaccharide (κ-carrageenan; supplier A vs. B). Similarly, formulations 5-3, 5-4, 5-5, and 5-6 differed only in the source of the polysaccharide (xanthan gum, supplier B, C, D, or E), but the erosion results varied considerably for each formulation, ranging from 3% to 100%. Furthermore, the results could not be explained from the specification sheets provided by the five different suppliers for each of the tested polysaccharide powders. Therefore, the characteristics and properties of each polysaccharide powder were investigated in an attempt to understand the contradictory results of the erosion test.

[0189] For each polysaccharide powder, different parameters were compared, including powder density, compressibility, Hausner ratio, angle of repose, cohesiveness, pH in solution, and viscosity (which is particularly related to polymer molecular weight). Furthermore, the literature reports that low salt concentrations promote interactions, while high salt concentrations tend to reduce or inhibit aggregation (Schmitt, 2000; de Kruif et al., 2004). Therefore, salt content was also compared based on the analytical certificates of each polysaccharide powder. These data are arranged in order of disintegration efficiency in Table 7.

[0190] Table 7: Characteristics of polysaccharide powders (5-1 to 5-6) classified based on D(%) / DT(min)

[0191]

[0192] * According to the supplier's analytical certificate.

[0193] The results shown in Table 7 reveal that tablet erosion in SGF(D / DT) cannot be reliably predicted from polysaccharide powder parameters such as pH, viscosity, salt concentration, tap density, or bulk density. In fact, most polysaccharide powders have very different salt concentrations. For example, although the polysaccharide powders of formulations 5-3 and 5-1 have the same salt content (4.3%), formulations 5-3 and 5-1 performed very differently in the erosion test (3% vs. 100%). These results are quite surprising given the well-known role of ionic forces and counter-charged ions in the formation of protein / polysaccharide complexes.

[0194] Interestingly, polysaccharide powders with higher Hausner ratios and Karl index / compressibility (indicating powder flowability) tend to form tablets that disintegrate more slowly (and vice versa). These results suggest that polysaccharide powders tend to aggregate with proteins to form sufficiently strong aggregates in situ, which should be cohesive (poor flowability), and conversely, particulate or sandy powders (e.g., without fine particles or fibers) have a lower ability to interact with protein powders after exposure to SGF, thus resisting erosion.

[0195] Angle of repose measurements showed that polysaccharide powders with higher angle values ​​were more likely to complex with protein powders after exposure to SGF, thus resisting erosion (and vice versa). Angle of repose values ​​indicate that cohesive powders are suitable for slowly disintegrating oral (or tablet / capsule) formulations, while free-flowing powders are not. This result is closely correlated with results obtained from the compressibility index and Hausner ratio values.

[0196] Finally, the angle of repose can be directly correlated with the dynamic cohesion index (Boschini et al., 2015). It appears that polysaccharide powders with a dynamic cohesion index below 10 cannot aggregate with proteins, while polysaccharide powders with a cohesion index above 10 readily interact with protein materials and form aggregates.

[0197] Interestingly, formulations with the lowest resistance to erosion were obtained using polysaccharide powders with a cohesive index below 5 and an angle of repose below 30 (e.g., see formulations 5-6 in Table 7, showing complete disintegration after only 12 minutes). Conversely, formulations with progressively increased resistance to erosion were obtained as the cohesive index and angle of repose of the polysaccharide powders increased (Table 7).

[0198] Table 8 summarizes the Hausner ratio, Karl index, angle of repose, dynamic cohesive index, and their corresponding disintegration time (if applicable) in SGF and disintegration percentage at the end of the gastric step for each polysaccharide powder tested. Overall, parameters related to the cohesiveness of the polysaccharide powder were observed to reliably predict the suitability of the polysaccharide powder for in-situ protein aggregate formation in the context of the oral delivery systems described herein. More specifically, polysaccharide powders with higher dynamic cohesive index, angle of repose, Hausner ratio, and Karl index (compressibility index) were observed to result in stronger in-situ aggregate formation after exposure to SGF, thereby providing stronger gastric protection of the active ingredient and / or slower release of the active ingredient.

[0199] Interestingly, for suitable polysaccharides, increasing their concentration in tablet formulations leads to increased disintegration time and a decrease in the percentage of disintegration at the end of the simulated gastric step (results not shown).

[0200] Table 8: Aggregate formation characteristics of polysaccharide powders based on D(%) / DT(min) classification

[0201]

[0202] Example 6:

[0203] Adjusting polysaccharide powder to increase aggregate formation and improve gastric protection

[0204] The results in Example 5 demonstrate that polysaccharide powders with higher cohesiveness (e.g., higher dynamic cohesive index, angle of repose, Hausner ratio, and Carr index) form stronger aggregates in situ upon exposure to SGF, thereby providing better gastric protection and / or slower release of the active ingredient. In Example 6, we demonstrate that, in the context of the oral delivery system described herein, polysaccharide powders with low cohesiveness and poor gastric protection can be modulated to increase cohesiveness, and this modulation results in significantly improved gastric protection.

[0205] Xanthan gum powder from supplier E (5-6) was chosen because it offered the least gastric protection (100% disintegration within 12 min) among all the polysaccharide powders shown in Table 8. We attempted to improve the physical properties of the xanthan gum powder from supplier E as follows: A 2% (w / v) solution was prepared in double-distilled water and subsequently lyophilized. After lyophilization, the powder was over-dried at 60°C for 5 days and then milled. After milling, the polysaccharide powder was sieved through a 20-mesh sieve. Table 9 illustrates the powder characteristics before and after adjustment. Tablets were then manufactured using each xanthan gum powder (raw and modified) as described in Table 5 and tested using a disintegration analyzer as described in Example 5. Table 10 illustrates the results obtained from each polysaccharide.

[0206] Table 9: Before and after adjustmentPolysaccharide properties

[0207]

[0208] As shown in Table 9, conditioning of xanthan gum powder resulted in a decrease in density (bulking and tapping) and a significant increase in parameters associated with greater powder cohesion (Hausner ratio, Karl index, angle of repose, and dynamic cohesion index). Conditioned xanthan gum powder also produced a significantly more fluffy powder. As shown in Table 10, formulations using conditioned xanthan gum powder resulted in tablets with very low disintegration rates (only 1% measurable) in a simulated gastric step.

[0209] Table 10: Tablet disintegration Solution parameters

[0210]

[0211]

[0212] Example 7:

[0213] Protein material types and their effects on tablet disintegration and probiotic survival

[0214] In this embodiment, we focus on the effect of protein type on the tablet release characteristics of different formulations. To this end, probiotic formulations containing *Lactobacillus helveticus* were prepared, comprising different protein source materials (Table 11). Six different protein materials from eight different suppliers were tested. For each protein type, the percentage of disintegration after SGF was measured after 60 min in a disintegrator (as described in Example 5). At the end of the gastric step, tablets were transferred to 750 mL of simulated intestinal fluid (SIF) consisting of 50 mM KH₂PO₄ buffer at pH 6.9 containing 0.5 g / L trypsin. For each tablet, disintegration was assessed and the disintegration time was measured. After disintegration, CFU was measured and compared with the initial count to evaluate cell viability percentage. All experiments were performed twice, and the results are shown in Table 12.

[0215] Table 11: Tested formulations

[0216]

[0217] Table 12: SGF post-disintegration percentage, disintegration time (SIF), and probiotic survival rate as a function of protein type

[0218]

[0219]

[0220] The results shown in Table 12 indicate that protein type and origin do not appear to have a significant effect on tablet disintegration in SGF. Cell viability is only slightly affected, and regardless of protein type or origin, *Lactobacillus helveticus* survival after SGF is shown to be between 40% and 55%, thus *Lactobacillus helveticus* is protected against the harsh conditions of the stomach. This effect is related to the buffering capacity of the protein material under these conditions. Furthermore, protein type appears to have an effect on the duration of simulated intestinal disintegration. Hemp and coconut proteins were observed to disintegrate more quickly, while rice and tara proteins were observed to disintegrate for longer periods. Pea protein showed a “moderate” disintegration time in simulated intestinal fluid. Without wishing to be bound by theory, these results can be explained—at least in part—by the fact that the composition of protein concentrates / isolates and the primary structure of proteins (e.g., the amount of hydrophobic amino acids and the amount of ionizable amino acids) can influence aggregate formation and its dissociation at pH 7.

[0221] Finally, the same formulation was tested by replacing *Lactobacillus helveticus* with other active ingredients (e.g., vitamin C, caffeine). Dissolution experiments also showed that these proteins are suitable for prolonging the in vitro release of the active ingredients through aggregation. Overall, these results indicate that aggregates can be formed using different protein materials and suppliers.

[0222] Example 8: Methods of Examples 9-22

[0223] 8.1 Preparation of tablets and capsules

[0224] Tablets were prepared by direct compression using a TDP-6 single-punch press (Yangzhou Nuoya Machinery Co., Ltd., Jiangju, China). The powder was weighed before compression and then mixed together in a mortar. The tablet diameter and thickness were 11.2 mm and 4.5–6.5 mm, respectively. 24 hours after manufacturing, tablet hardness was measured using a YD-1 tablet hardness tester (Minsheng Pharmaceutical Machinery Ltd., Shanghai, China) according to USP standards. <1217> Measure tablet hardness. Hardness is between 5 and 9 kp. Each polysaccharide powder used has an angle of repose greater than 35°, a Hausner ratio greater than 1.18, a Karl index / compressibility index greater than 15, and a dynamic cohesion index greater than 10.

[0225] In the case of capsule formulations, once the powder is mixed, capsules are filled using a manual capsule filling machine. All tests were performed using “vegetarian” (HMPC) capsules “0”.

[0226] 8.2 Dissolution characteristics of tablets / capsules

[0227] The in vitro properties of tablet / capsule formulations were tested using an SR6 dissolution tester at USPII (Hanson Research Corp., Chatsworth, CA). This was conducted in accordance with USP... <2040> The guidelines described in [the document] (disintegration and dissolution of dietary supplements).

[0228] During the experiment, the paddle speed was set to 60-100 rpm, and the temperature was maintained at 37°C. Before measurement, the sample was filtered, and background absorbance was subtracted. After release, the concentration of the active ingredient (AI) dissolved in the release medium was measured as a function of time. Depending on the active ingredient, release measurements were performed by HPLC or directly by spectrophotometry.

[0229] A typical dissolution test consists of immersing three tablets in simulated gastric fluid (SGF) for 2 hours, followed by immersion in simulated intestinal fluid (SIF) until complete dissolution. In the case of capsule formulations, a stainless steel settling device with six spiral components is used to ensure immersion.

[0230] SGF consisted of a diluted HCl (37%) solution containing 2 g / L NaCl and 0.1 g / L pepsin, pH 1.0. SIF consisted of a buffer solution (50 mM) containing 0.5 g / L trypsin and NaH₂PO₄, pH 6.9. Experiments were repeated at least twice.

[0231] 8.3 Tablet disintegration characteristics

[0232] Tablet disintegration was tested using a disintegration analyzer (Tianjin Guoming Medicinal Equipment Co., Tianjin, China). The test was conducted in accordance with USP specifications. <2040> (Disintegration and dissolution of dietary supplements) were tested. In a typical experiment, one tablet was placed in each of three test tubes in a basket. No settler was used. SGF was used as the immersion solution at 37°C, and the apparatus was run for 1 hour. The tablet was then transferred to SIF until complete disintegration. Tablet disintegration was determined by gravimetric analysis. The experiment was repeated at least twice.

[0233] 8.4 Probiotic survival

[0234] When the active ingredient (AI) is a probiotic or a mixture of probiotics, postgastric cell survival is of particular concern. Therefore, microbial counting was performed at the end of the disintegration assay. The strains were cultured according to appropriate procedures.

[0235] In the case of yeast, strains were cultured using YPD agar. All samples were cultured under aerobic conditions and incubated at 30°C for 48–72 h.

[0236] In the case of lactobacillus / bifidobacterial strains, MRS agar was used to culture the strains. All samples were cultured under anaerobic conditions and incubated at 37°C for 48–72 h.

[0237] 8.5 Protein hydrolysis activity

[0238] The pancreatic enzyme proteolytic activity (%) was measured as follows: At the end of the simulated stomach step, the pH was adjusted to 7-7.5, the tablet formulation was disintegrated using a rotor-stator homogenizer, and 500 mg of native whey protein was added. After incubation at 37°C for 4 hours, proteolysis was measured by quantifying free amino groups using OPA (o-phenylenedialdehyde) reagent. The same amount of unencapsulated / unprotected pancreatic enzyme was used, and experiments were conducted with or without a simulated stomach passage (negative control). The proteolytic activity (%) was determined by calculating the ratio of the free amino group concentration of a given tablet formulation to that of the positive control.

[0239] Example 9: Probiotic tablet formulation and release

[0240] Postgastric survival rate and survival rate of probiotic strains using the formulations described in Table 13 Figure 8A The survival rate of unprepared strains was shown in Figure 8B The probiotics used were a mixture of *Lactobacillus paracasei*, *Lactobacillus rhamnosus*, and *Lactobacillus plantarum*. The protein:polysaccharide mixture had a weight ratio of 90:10. The disintegration kinetics of the formulation described in Table 13 are shown in... Figure 9 middle.

[0241] Table 13: Preparations using three probiotic strains

[0242]

[0243] Example 10: Curcumin tablet formulation and release

[0244] The dissolution profiles obtained from the formulations described in Table 14 are shown in... Figure 10 The ratio of protein to polysaccharide mixture is 92:8.

[0245] Table 14: Curcumin Tablet Formulations

[0246]

[0247] Example 11: 5-HTP tablet formulation and release

[0248] The dissolution profiles obtained from the formulations described in Table 15 are shown in... Figure 11 The ratio of protein to polysaccharide mixture is 85:15.

[0249] Table 15: 5-HTP Tablet Formulations

[0250]

[0251]

[0252] Example 12: Peppermint extract tablet formulation and release

[0253] The dissolution characteristics of the formulations described in Table 16 are shown in... Figure 12 The ratio of protein to polysaccharide mixture is 70:30.

[0254] Table 16: Peppermint Extract Tablet Formulations

[0255]

[0256] Example 13: Coffee tablet formulation and release

[0257] The dissolution characteristics of the formulations described in Table 17 are shown in... Figure 13 The ratio of protein to polysaccharide mixture is 92:8.

[0258] Table 17: Caffeine Tablet Formulations

[0259]

[0260]

[0261] Example 14: Formulation and release of Solidago virgaurea extract / ginger tablets

[0262] The dissolution characteristics of the formulations described in Table 18 are shown in... Figure 14 The ratio of protein to polysaccharide mixture is 75:25.

[0263] Table 18: One tablet formulation of daylily & ginger

[0264]

[0265] Example 15: Saccharomyces boulardii tablet formulation and release

[0266] Survival of *Saccharomyces boulardii* after gastric ingestion. Survival rates of strains using the formulations described in Table 19 are shown in... Figure 15A (76.1%) and the survival rate of the unprepared strains are shown in Figure 15B (3%). The disintegration kinetics of the formulations described in Table 19 are shown in... Figure 16The ratio of protein to polysaccharide mixture is 85:15.

[0267] Table 19: Saccharomyces boulardii tablet formulations

[0268]

[0269]

[0270] Example 16: β-Alanine Tablet Formulation and Release

[0271] The disintegration kinetics of the formulations described in Table 20 are shown in... Figure 17 The ratio of protein to polysaccharide mixture is 85:15.

[0272] Table 20: β-Alanine Tablet Formulations

[0273]

[0274] Example 17: Peppermint-Vitamin B6 Capsule Formulation and Release

[0275] The dissolution characteristics of the formulations described in Table 21 are shown in... Figure 18 The ratio of protein:xanthan gum:polysaccharide mixture is 80:10:10.

[0276] Table 21: Peppermint Capsule Formulations

[0277]

[0278]

[0279] Example 18: Melatonin-ginger tablet formulation and release

[0280] The dissolution characteristics of the formulations described in Table 22 are shown in Figure 19 The ratio of protein to polysaccharide in the mixture is 80:20.

[0281] Table 22: Melatonin-Ginger Tablet Formulation

[0282]

[0283] Example 19: Formulation and Release of Nicotinamide Mononucleotide (NMN) Tablets

[0284] The dissolution characteristics of the formulations described in Table 23 are shown in Figure 20 The ratio of protein:xanthan gum:guar gum mixture is 78:18:4.

[0285] Table 23: NMN Tablet Formulations

[0286]

[0287] Example 20: Vitamin C capsule formulation and release

[0288] The dissolution characteristics of the formulations described in Table 24 are shown in Figure 21 The ratio of protein to polysaccharide in the mixture is 78:22.

[0289] Table 24: Vitamin C Capsule Formulations

[0290]

[0291] Example 21: Pancreatic enzyme tablet formulation and release

[0292] The dissolution characteristics of the formulations described in Table 25 are shown in... Figure 22 The ratio of protein:xanthan gum:algin mixture was 80:16:4. The trypsin proteolytic activity after SGF treatment, compared with the negative control, was shown in... Figure 23 middle.

[0293] Table 25: Pancreatic Enzyme Tablet Formulations

[0294]

[0295] Example 22: Garlic tablet formulation and release

[0296] The dissolution characteristics of the formulations described in Table 26 are shown in Figure 24 The protein:polysaccharide ratio is 86:14.

[0297] Table 26: Garlic Tablet Formulations

[0298]

[0299] References

[0300] Beakawi Al-Hashemi,HM,Baghabra Al-Amoudi,OS(2018).A review on the angle of repose of granular materials.Powder technology,330,397-417.

[0301] Boschini,F.,Delaval,V.,Traina,K.,Vandewalle,N.,Lumay,G.(2015).Linkingflowability and granulometry of lactose powders.International Journal ofPharmaceutics,494,312-320.

[0302] Carr,R.L.,(1965)Evaluating Flow Properties of Solids.Chem.Eng.72,163-168.

[0303] de Kruif,C.G.,Weinbreck,F.,de Vries,R.(2004).Complex coacervation ofproteins and anionic polysaccharides.Current opinion in colloid and interfacescience,9,340-349.

[0304] Gilbert,V.,Rouabhia,M.,Wang,H.,Amould,A-L.,Remondetto,G.&Subirade,M.(2005).Characterization and evaluation of whey proteins-based films assubstrates for in vitro cell cultures.Biomaterials,26,7471-7480.

[0305] Schmitt,C.(2000).Etude de la coacervation complexe entre la P-lactoglobuline et la gomme d’acacia en solution aqueuse.These INSAIA,France.217pp.

[0306] Syrbe,A.,Bauer,W.J.&Klostermeyer,H.(1998).Polymer science concepts indairy systems-An overview of milk protein and food hydrocolloidinteraction.International Dairy Journal,8,179-193.

[0307] Tolstoguzov,V.B.(1997).Protein-polysaccharide interactions.In:S.Damodaran&A.Paraf,Food 5 Proteins and their Applications(ppl71-198).NewYork:Marcel Dekker。

Claims

1. An oral delivery system comprising a dried homogeneous mixture including a mixture of protein powder and polysaccharide powder and an active ingredient dispersed therein, wherein, upon immersion of the oral delivery system in gastric juice, the protein powder and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ, thereby imparting gastric protection to the active ingredient and / or improved release of the active ingredient, wherein, The ratio of protein powder to polysaccharide powder in the oral delivery system is altered to change the level of gastric protection of the active ingredient and / or the release rate of the active ingredient, wherein the polysaccharide powder has or is adjusted to have one or more of the following powder flow characteristics: (a) The angle of repose is greater than 40 degrees; (b) The dynamic cohesion index is greater than 14; (c) The Carr compressibility index is greater than 23%; (d) Hausner ratio greater than 1.28; In the oral delivery system, the weight ratio of the polysaccharide powder to the protein powder is 1:20 to 1:

1.

2. The oral delivery system according to claim 1, wherein, The polysaccharide powder has or is adjusted to have all the powder flow characteristics defined in (a), (b), (c) and (d).

3. The oral delivery system according to claim 1 or 2, wherein, After immersing the oral delivery system in a solution with a pH lower than the pKa of the polysaccharide, the protein powder and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ.

4. The oral delivery system according to claim 1 or 2, wherein: (a) Increasing the ratio of polysaccharide powder to protein powder in the oral delivery system to increase the gastric protection level of the active ingredient and / or decrease the release rate of the active ingredient; (b) The weight ratio of polysaccharide powder to protein powder in the oral delivery system is 1:15 to 1:1.5; (c) The oral delivery system comprises 5% to 50% w / w of the protein powder and polysaccharide powder mixture.

5. The oral delivery system according to claim 1 or 2, wherein: (a) After the oral delivery system is immersed in simulated gastric fluid, the protein powder and polysaccharide powder mixture forms a protein / polysaccharide complex aggregate in situ, wherein the simulated gastric fluid consists of a 37% v / v diluted HCl solution at pH 1.0; the HCl solution contains 2 g / L NaCl and 0.1 g / L pepsin. (b) The release rate of the active ingredient is inversely proportional to the strength of the protein / polysaccharide complex aggregate formed in situ after the oral delivery system is immersed in the gastric juice; or (c) The protein / polysaccharide complex aggregate formed in situ after the oral delivery system is immersed in simulated gastric fluid is characterized by the presence of intramolecular β-sheets, α-helices and / or disordered structures, wherein the simulated gastric fluid consists of a 37% v / v diluted HCl solution at pH 1.0; the HCl solution contains 2 g / L NaCl and 0.1 g / L pepsin.

6. The oral delivery system according to claim 1 or 2, wherein the oral delivery system is: (a) Delayed-release oral delivery system; (b) a prolonged-release oral delivery system; or (c) An oral delivery system that provides enhanced gastric protection for the active ingredient after oral administration, compared to administering the active ingredient alone.

7. The oral delivery system according to claim 6, wherein: (a) Compared to corresponding oral delivery systems lacking the polysaccharide powder, the delayed-release oral delivery system delays the time required for the release of 50% of the active ingredient by at least 30 minutes; (b) The extended-release oral delivery system results in the release of the active ingredient for a period of at least 3 hours; and / or (c) Compared to administering the active ingredient alone, the oral delivery system provides enhanced gastric protection for the active ingredient; The above results are based on dissolution tests, including immersing the oral delivery system in simulated gastric fluid at 37°C for 2 hours, followed by immersion in simulated intestinal fluid. The simulated gastric fluid consists of a 37% v / v diluted HCl solution at pH 1.0 containing 2 g / L NaCl and 0.1 g / L pepsin; and the simulated intestinal fluid consists of a 50 mM NaH2PO4 or KH2PO4 buffer solution at pH 6.9 containing 0.5 g / L pancreatin.

8. The oral delivery system according to claim 1 or 2, wherein, The protein powder includes: natural proteins; denatured proteins; or mixtures thereof.

9. The oral delivery system according to claim 1 or 2, wherein, The protein powder includes: unmodified protein; chemically modified protein; or mixtures thereof.

10. The oral delivery system according to claim 1 or 2, wherein, The protein powder includes: plant protein; animal protein; or mixtures thereof.

11. The oral delivery system according to claim 1 or 2, wherein, The polysaccharide powder includes: unmodified polysaccharides; chemically modified polysaccharides; or mixtures thereof.

12. The oral delivery system according to claim 1 or 2, wherein, The polysaccharide powder includes: polysaccharides containing negatively charged polysaccharides and / or acidic groups.

13. The oral delivery system according to claim 1 or 2, wherein, The polysaccharide powder includes: carrageenan, xanthan gum, alginate, pectin powder, agar, gellan gum, guar gum, carboxymethyl cellulose, sophora bean gum, mannan, glucomannan, hyaluronic acid, tamarind gum, psyllium husk gum, tara gum, acacia gum, gum arabic, gat gum, astragalus gum, ebony gum, cinnamon gum, Brunei gum, gel polysaccharide, fucoidan, or any mixture thereof.

14. The oral delivery system according to claim 1 or 2, wherein, The active ingredient is a dietary supplement or a drug.

15. The oral delivery system according to claim 1 or 2, wherein, The active ingredient is a food extract or herbal supplement.

16. The oral delivery system according to claim 1 or 2, wherein, The active ingredient is probiotics, vitamins, or amino acids.

17. The oral delivery system according to claim 1 or 2, further comprising one or more nutritionally or pharmaceutically acceptable excipients and / or additives.

18. The oral delivery system according to claim 17, wherein, The additives include the following: Microcrystalline cellulose, magnesium stearate and / or silicon dioxide.

19. The oral delivery system according to claim 1 or 2, wherein the oral delivery system is a tablet or capsule.

20. The oral delivery system according to claim 1 or 2, wherein, The oral delivery system does not include enteric coating.

21. A method for preparing a solid oral dosage form, the method comprising dispersing an active ingredient in a dry, homogeneous mixture comprising protein powder and polysaccharide powder, and formulating the resulting mixture into a solid oral dosage form, wherein the polysaccharide powder has powder flow characteristics and is capable of interacting with the protein powder such that immersion of the solid oral dosage form in gastric juice results in the in-situ formation of a protein / polysaccharide complex aggregate, thereby imparting gastric protection to the active ingredient and / or improved release of the active ingredient, wherein... The polysaccharide powder has one or more of the powder flow characteristics defined according to claim 1; wherein, the weight ratio of the polysaccharide powder to the protein powder in the solid oral dosage form is 1:20 to 1:

1.

22. The method according to claim 21, wherein, The solid oral dosage form is an oral delivery system as defined in any one of claims 1 to 20.

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