Novel formulation for oral administration of therapeutic agents to the digestive tract

Abstract

Novel nanocomposites for oral administration for digestive diseases (e.g., IBD) or digestive injuries are described herein. The nanocomposites include nanoemulsion particles (NE) filled with an active therapeutic agent, and those nanoemulsion particles (NE) are then combined with a second component (optionally in powder form) that includes a self-assembling peptide (SAP) or a peptidomimetic. When administered and hydrated at a pH near slightly acidic or neutral, the system self-assembles into a hydrogel matrix that encapsulates the NE. The hydrogel matrix then adheres to the intestinal tract of the subject being treated and delivers the active agent to that intestinal tract more efficiently. The nanocomposite can be administered in a capsule dosage form that forms a depot in the stomach or after the stomach.

Description

Technical Field

[0001] (Priority) This application claims the priority of U.S. Provisional Patent Application No. 63 / 342,081, filed on May 14, 2022, and U.S. Provisional Patent Application No. 63 / 412,464, filed on October 2, 2022, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0002] (Sequence Listing) This application includes an XML sequence listing submitted electronically, which is hereby incorporated by reference in its entirety. This sequence listing, created on April 10, 2023, is named 3DM-21-06-ORAL-PCT_SL.xml and is 31,763 bytes in size.

[0003] (Field of the Invention) The present disclosure relates to methods of making and using pharmaceutical formulations for the treatment of gastrointestinal diseases or injuries.

Background Art

[0004] (Background) Inflammatory bowel disease (IBD) often requires long-term treatment to promote mucosal healing and induce and maintain long-term clinical remission. Treatment options include corticosteroids, 5-aminosalicylic acid (5-ASA), immunomodulators, immunosuppressants, and / or therapeutics. Although numerous treatment options are available, many can induce complications related to the type of drug, and patients may become intolerant to treatment over time (Lefevre, P.L.C. and Casteele, N. Vande, Clinical pharmacology of janus kinase inhibitors in inflammatory bowel disease, Journal of Crohn’s and Colitis, Vol. 14, S725–S736 (2020)). Understanding of the involvement of inflammatory cytokines in the etiology of inflammatory bowel disease has improved, and Janus kinase (JAK) inhibitors have emerged as an effective oral treatment option for ulcerative colitis. By inhibiting the cytokine-activated JAK components of the JAK-signal transducer and activator of transcription (STAT) pathway, JAK inhibitors interfere with the signaling of various cytokines involved in the abnormal immune response that contributes to the development of IBD. (Zundler, S. and Neurath, M.F., Integrating immunologic signaling networks: The JAK / STAT pathway in colitis and colitis-associated cancer, Vaccines, Vol. 4 (2016). Tofacitinib (Xeljanz®) is a first-in-class small molecule JAK inhibitor approved for the treatment of moderate to severe ulcerative colitis in Europe and the United States.

[0005] Oral administration of therapeutic agents is a preferred route, improving safety, convenience, and patient compliance. Furthermore, the oral route facilitates local drug delivery and systemic accumulation in the gastrointestinal tract (GIT). However, the oral bioavailability of many drugs is limited by various biological barriers. In an attempt to enhance therapeutic activity (especially for local applications), various hybrid systems have been developed that result in a prolonged drug residence time in the gastrointestinal tract, thereby leading to an increase in drug delivery, as described, for example, in Sharma, S. and Sinha, V.R., Current pharmaceutical strategies for efficient site specific delivery in inflamed distal intestinal mucosa, Journal of Controlled Release, Vol. 272, 97 - 106 (2018), including, for example, polymethacrylate delivery systems, carbohydrate - based delivery systems, and vesicular systems. In a study previously reported by Rosso et al., Control. Release (2021) 333:579 - 592, nanocomposite sponges based on the naturally occurring polysaccharide (i.e., chitosan) were evaluated for enhancement of intestinal residence time after oral administration.

[0006] More recently, drug - carrying nanoparticles have increasingly been combined with hydrogels. These hydrogels are usually made from polymers (e.g., the above - mentioned chitosan, alginic acid, dextran, carrageenan, polycaprolactone (PCL), and hydroxypropylmethylcellulose (HPMC)) to form hybrid systems for controlled or enhanced drug delivery to the intestine (Andretto, V., Rosso, A., Briancon, S. and Lollo, G., Nanocomposite systems for precise oral delivery of drugs and biologics, Drug Deliv.Transl.Res., 11, 445 - 470 (2021)).

[0007] Hydrogels represent the most used type of nanocomposites in oral delivery at the macroscale and microscale. However, ingestible soft hydrogels can face practical problems, particularly pH-dependent behavior, mechanical weakness, and excessive swelling rates, which shorten their gastrointestinal (GI) residence time and limit storage stability. The use of nanocomposites as multiple-compartment capsules, tablets, aerogels, sponges, and films has been described. The main requirement for such solid systems is that both the nanoparticles and the polymer matrix must recover their initial properties and recognize specific target sites to exert their activity after reconstitution in the GI medium. This involves careful selection of the components of these nanocomposites, paying attention to their FDA approval for the oral route, physicochemical properties, structural properties (crystallinity, fluidity), and the interactions between them. Another important aspect is the understanding and prediction of nanoparticle release from the nanocomposite. The main strategies described are based on pH-induced matrix degradation or dissolution, chemically or enzymatically driven matrix erosion, pH- or temperature-dependent swelling of the polymer network, and diffusion or desorption of the nanoparticles / drugs. For a better understanding of their release kinetics, the effective diffusion coefficients within various polymer matrices should be estimated by modeling the diffusion process.

[0008] Therefore, the need remains to develop further drug delivery systems for the improved treatment of digestive diseases or injuries.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Non - Patent Document 4

Non - Patent Document 5

Summary of the Invention

Means for Solving the Problems

[0010] (Summary of the Invention) A hybrid system composed of muco-permeable liquid nanoparticles (NEs) embedded in a self-assembling peptide hydrogel has been developed. The self-assembling peptide is described, for example, in U.S. Patent No. 9,724,448, which is incorporated herein by reference. The self-assembling peptide RADARADARADARADA (SEQ ID NO: 1) (also known as RADA16) (a "2.5% v / w product containing RADA16, called PURASTAT" (also referred to herein as "PS" or "PM" and used interchangeably), available from 3-D Matrix, Ltd.; www.3dmatrix.com) was selected as the model self-assembling peptide and used at the indicated fractional concentration. Tofacitinib (TFC) was selected as the model drug for proof of concept, but any other hydrophobic small molecule drug or any other suitable drug may be used. In another embodiment of the present invention, the drug is budesonide, a corticosteroid-like hydrophobic drug. For example, other small molecule drugs listed in the publication by Fitzpatrick et al. (e.g., S1P receptor modulators, other JAK inhibitors, CCR9 antagonists, α4 integrin antagonists, immunomodulators) may be ideally encapsulated in the nanosystem for the preparation of various nanocomposites (Fitzpatrick, L.R. and T. Woldemariam "Small-molecule drugs for the treatment of inflammatory bowel disease" (2017): 495-510). The hybrid system can transport and release the nanosystem filled with the selected muco-permeable drug to the intestinal wall, thereby maximizing the local drug effective amount by controlling the permeability of the drug and enhancing the biological stability of the drug, as well as providing rapid induction of hemostasis by the hydrogel.

[0011] The use of the hybrid system composed of PS hydrogel has at least two distinct advantages: a long residence time in the gastrointestinal tract (GIT) resulting in increased drug delivery, and enhanced epithelial and tissue regeneration due to the properties of the hydrogel, which are similar to those of the extracellular matrix.

[0012] Furthermore, specific disease factors were improved by administration of the PS hydrogel / NE mixture alone in the absence of any active ingredients.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19A

Figure 19B

Figure 19C

Mode for Carrying Out the Invention

[0014] (Detailed Description of the Invention) After the development and characterization of the following systems, a generally recognized mouse model for in vivo studies of treatments for etiologies involved in intestinal inflammation was utilized as proof of the effectiveness of a nanocomposite system for desirable applications in the area of chronic inflammation of the gastrointestinal tract (GIT), particularly related to the family of diseases grouped under the general term of inflammatory bowel disease (IBD). The use of a hybrid system containing the PURASTAT (PM; PS, RADA16 (SEQ ID NO: 1)) hydrogel, and more generally other suitable self-assembling peptides (SAPs), has the following numerous additional advantages: long residence time in the GIT, increased local drug delivery by providing a double encapsulation strategy, and epithelial and tissue regeneration promoted by the hydrogel itself. In fact, treatment with PURASTAT has also been shown to result in rapid hemostasis of leaking wounds and a reduction in delayed bleeding that do not rely on the natural coagulation process in other applications (references) (e.g., surgical interventions), even when the patient's natural hemostatic mechanism is impaired or dysfunctional.

[0015] A further advantage of this nanocomposite system is that it can be dried and then reconstituted without substantial change in its properties under in vivo or in vivo-like conditions. The dried nanocomposite offers the advantage of long-term storage, but perhaps more importantly, the dried nanocomposite powder can be encapsulated in various types of capsules known in the art for controlled local delivery in the precise part of the GIT using systems differentiated based on GIT transit time, pH changes, bacterial enzymes, mucoadhesion, nanotechnology, and disease-related triggers. One such capsule, in which a hydrated RADA16 hydrogel doped with the polymeric dye fluorescein isothiocyanate-dextran 4 kDa (FD4), was designed for release in the large intestine and is described by Eleftheriadis et al. (Pharm. Development Tech. 25(4):1-23(2020)). The reference discloses pH-sensitive hydroxypropylmethylcellulose phthalate (HPMCP)-based 3D printed capsules. Other encapsulating materials suitable for the present invention include poly(meth)acrylate polymers and members of the Eudragit family (Chu, J.N. and Traverso, G., Foundations of gastrointestinal-based drug delivery and future developments, Nature Reviews: Gastroenterology & Hepatology (2021) doi:10.1038 / s41575-021-00539-w).

[0016] The present invention is based on at least the following findings / parameters: 1. Nanocomposites based on a physical mixture of a pre-formed nanosystem (i.e., nanoemulsion, NE) filled with a drug for the treatment of IBD and a pre-formed self-assembling peptide (BASED)-based hydrogel (PURASTAT) were made with various concentrations of SAP. 2. The preparation of nanocomposites characterized by two different concentrations led to the development of systems with different structural properties, resulting in differences in their drug release behavior. 3. The presence of the nanosystem did not change the secondary structure of the peptide, which was very important for its hydrogel formation and the maintenance of its structure. 4. The use of the nanocomposite to test the therapeutic efficacy of the drug against the IBD mouse model led to results based on various techniques detailed herein. 5. Certain disease factors were improved by the administration of the PS hydrogel / NE mixture alone, even in the absence of any active ingredient.

[0017] Disease Activity Index (DAI) score (which is used to evaluate DSS-induced colitis). Daily, animals were observed for weight loss, stool consistency, and the presence of gross hemoccult and bloody anus. For each parameter, a score of 0 - 4 was assigned and then divided by 3 to yield a total DAI score in the range of 0 (non-diseased) - 4 (severe colitis) (Goncalves, F. da C., Schneider, N., H.M.-A.S. & 2013, U., Characterization of Acute Murine Dextran Sodium Sulfate (DSS) Colitis: Severity of Inflammation is Dependent on the DSS Molecular Weight and Concentration, Acta Sci. Vet. 41, 1 - 9 (2013)). Colon length and weight (which often decrease in cases of inflammation) (Biton, I.E. et al., Assessing Mucosal Inflammation in a DSS-Induced Colitis Mouse Model by MR Colonography, Tomogr. (Ann Arbor, Mich., 4, 4 - 13 (2018)). Activity of the enzyme myeloperoxidase (MPO) (which is a direct indicator of neutrophil infiltration into the colonic mucosa and thus an important marker of inflammation) (Hansberry, D.R., Shah, K., Agarwal, P. and Agarwal, N., Fecal Myeloperoxidase as a Biomarker for Inflammatory Bowel Disease, Cureus, 9 (2017)). Histomorphological evaluation by HPS staining (hematoxylin - phloxin - safranin staining) similar to H&E. HPS can distinguish between the most common connective tissue (collagen) and muscle and cytoplasm by staining the former yellow and the latter two pink, unlike H&E staining which stains all three pink. Immunohistochemical examination for the detection of CD45-positive cells.

[0018] (Formulation and Characterization of Nanoemulsion (NE) Particles) The composition, formulation, and characterization of nanoemulsion particles as components of the present invention have been mostly previously described by Rosso et al. (Development and structural characterization of a novel nanoemulsion for oral drug delivery, Colloids and Surfaces A (2020) 593:124614). As described in that reference, the NE was prepared by emulsion phase inversion (EPI) technology combined with high stirring energy input. Briefly, the NE is composed of a middle-chain triglyceride (MCT) (Miglyol® 812) oil core purchased from CREMER OLEO GmbH & Co. KG (Hamburg, Germany), stabilized by a surfactant shell made of a mixture of a hydrophilic surfactant (i.e., polyoxyethylene (40) stearate (Myrj® 52), identified herein as S1) and a hydrophobic surfactant (i.e., oleoylpoli-oxy-6 glyceride (Labrafil® M1944CS), identified as S2). To prepare the oil phase, MCT (0.35 g) and surfactant (1 g) were mixed and magnetically stirred (750 rpm) using a thermostatic bath at 80 °C. The aqueous phase (5 mM PBS, 3.65 mL), also heated to 80 °C, was added to the organic melt phase. Then, stirring was carried out by two cycles of 10 minutes using a rotor-stator disperser (T25 digital Ultra-Turrax® (Werke GmbH & Co. KG, Staufen, Germany) equipped with an S25N-10G shaft IKA®) rotating at 11,000 rpm at 80 °C.

[0019] As described herein, medium-chain triglycerides (MCT) (Miglyol® 812), purchased from CREMER OLEO GmbH & Co. KG (Hamburg, Germany), were used as the oil forming the NE core. Polyoxyethylene (40) stearate ((Myrj® 52), identified herein as S1 and obtained from Sigma-Aldrich (St Quentin-Fallavier, France)) and oleoylpoli-oxy-6 glyceride ((Labrafil® M1944CS), identified herein as S2 and obtained from Gattefosse (Saint-Priest, France)) were used as non-ionic surfactants constituting the outer shell of the NE. The aqueous phase used to prepare the emulsion was a sodium phosphate buffer solution (5 mM; pH 7.4). Tacrolimus, a hydrophobic model drug selected to be encapsulated in the NE in these studies and a BCS class II immunomodulatory agent used in the treatment of various diseases, was purchased from LC Laboratories (Woburn, MA, USA).

[0020] The materials selected and their ratios were the result of a series of experiments described by Rosso et al. (2020). To investigate the NE region, three ternary phase diagrams were designed using 23 formulations for each diagram. These three-component mixtures were composed of oil, water, and three different surfactant mixtures (S1 + S2) called Smix. Smix was characterized by the surfactant mass ratio (SMR) of S1 to S2 SMR = mass of S1 / mass of S2 Equation (1).

[0021] The NE region was identified by varying the amounts of Smix / oil / water at fixed SMRs of 1, 2, 5, and 5. Another parameter of the formulation was SOR = mass of Smix / mass of MCT Equation (2) the surfactant-to-oil ratio (SOR) defined as such.

[0022] In the reported studies, various NEs configured as described immediately above were comprehensively characterized by methods known in the art according to electrical conductivity, shear viscosity, X-ray powder diffraction (XRPD) analysis, size distribution, surface potential, morphology, and other characteristics. Based on the analysis of the results of these experiments, further experiments and analyses were conducted to select NE compositions for further use. The optimized NE had an average droplet diameter of about 104 ± 3 nm, a low polydispersity index (PDI) of (0.2), and a neutral / slightly negative zeta potential of (-9 ± 1 mV). This neutral surface charge, derived from the PEGylated surfactant (S1) shell, prevents interaction with the intestinal contents and mucosal layer, thereby enhancing the diffusion of the NE across the epithelium and its translocation through the mucosa (Hua et al., Advances in oral nano-delivery systems for colon targeted drug delivery in inflammatory bowel disease: selective targeting to diseased versus healthy tissue, Nanomed. Nanotechnol. Biol. Med. 11 (2015) 1117 - 1132; doi.org / 10.1016 / j.nano.2015.02.018).

[0023] As an example of further use of the optimized NE reported by Rosso et al. (2020), tacrolimus (a BCS class II immunomodulatory agent used in the treatment of various diseases) (Zhang et al., Multifunctional Poly(methyl vinyl ether-comaleic anhydride)-graft-hydroxypropyl-β-cyclodextrin amphiphilic copolymer as an oral high-performance delivery carrier of tacrolimus, Mol. Pharm. 12 (2015) 2337-2351; doi.org / 10.1021 / acs.molpharmaceut.5b00010) was encapsulated in NE as a hydrophobic model drug. The tacrolimus-loaded NE was observed to be relatively stable for 28 days during storage at 20 °C and 37 °C, and showed a delayed release of the drug compared to the drug dissolved in solution in vitro in a mimicked GI environment.

[0024] The tofacitinib-loaded NE used in the in vivo efficacy study disclosed below was prepared by dissolving the drug in the organic phase to reach a final concentration of 4 mg / mL. The NE loaded with DiD (fluorescent label) was prepared by adding a carbocyanine derivative fluorescent dye to the organic phase to obtain a final concentration of 0.057 mg / mL for the in vivo distribution in vivo study disclosed in this patent application. Table 1 shows the characteristics of the NE prepared by the method described by Rosso et al. (2020). [Table 1]

[0025] The size distribution and surface potential of NE droplets were determined using a Malvern Zetasizer® Nano ZS instrument (Malvern Instruments S.A., Worcestershire, UK). The particle size was measured by dynamic light scattering (DLS) at a scattering angle of 173° at 25 °C. The ζ potential was calculated from the average electrophoretic mobility measured for samples diluted in milliQ water. The stability of the blank and TFC-NE in the colloidal suspension was followed for 6 months at 4 °C. At the planned time points, the particle size, polydispersity index (PDI), and ζ potential were measured.

[0026] (General rheological and structural characteristics of PURASTAT filled with nanoemulsion particles (NE) to form nanocomposites) For the formulation of nanocomposites, nanoemulsion (NE) was efficiently loaded into PURASTAT (SEQ ID NO: 1) at different SAP concentrations of 0.5% w / v and 2.0% w / v. (It should be noted that when referring to the amount of SAP or peptidomimetic (collectively referred to as "peptoproduct") in a solution (sometimes vaguely called "hydrogel" depending on the nature of its final product and at the same time implying "solution"), "v / w" refers to the weight of that which is substantially pure (e.g., at least 65%, at least 70%, preferably at least 75%, at least 80%, at least 85%, most preferably at least 90%, at least 95%, or purer) compared to the full-length "peptoproduct" in the solution before hydrogel formation.

[0027] NE filled with tofacitinib (TFC) was prepared by dissolving the drug in the organic phase such that it reached the desired final concentration of 4 mg / mL for in vivo studies using nanocomposites. NE filled with DiD was prepared by adding this carbocyanine derivative fluorescent dye in the organic phase to obtain final concentrations of 0.5 mg / mL for in vitro assays and 0.057 mg / mL for in vivo biodistribution studies.

[0028] IBD often requires long-term treatment to promote mucosal healing and induce and maintain long-term clinical remission. Such treatment includes the use of corticosteroids, 5-aminosalicylic acid (5-ASA), immunomodulators, immunosuppressants, and / or targeted biological agents (e.g., monoclonal antibodies). Although numerous treatment options are available, many can induce complications related to the type of drug, and patients may become intolerant to the treatment over time (Pavine L C Lefevre, Niels Vande Casteele, Clinical Pharmacology of Janus Kinase Inhibitors in Inflammatory Bowel Disease, Journal of Crohn’s and Colitis, 14, Suppl 2, July 2020, S725–S736, https: / / doi.org / 10.1093 / ecco-jcc / jjaa014). Understanding of the involvement of inflammatory cytokines in the etiology of inflammatory bowel disease has improved, and Janus kinase (JAK) inhibitors have emerged as an effective oral treatment option for ulcerative colitis. By inhibiting the cytokine-activated JAK components of the JAK-signal transducer and activator of transcription (STAT) pathway, JAK inhibitors modulate the abnormal immune response mediated by cytokines, which contribute to the development of IBD. [See also Zundler, S.; Neurath, M.F., Integrating Immunologic Signaling Networks: The JAK / STAT Pathway in Colitis and Colitis-Associated Cancer, Vaccines, 2016, 4, 5.; doi.org / 10.3390 / vaccines4010005]. Tofacitinib (Xeljanz®) is a first-in-class small molecule JAK inhibitor approved for the treatment of moderate to severe ulcerative colitis in Europe and the United States. For these reasons, tofacitinib (TFC) was selected as the model drug for the proof of concept of the inventors' project. In another embodiment of the present invention, the drug is budesonide, a cortisone-like hydrophobic drug.For example, other small molecule drugs listed in the Fitzpatrick et al. publication (e.g., S1P receptor modulators, other JAK inhibitors, CCR9 antagonists, α4 integrin antagonists, immunomodulators) can be ideally encapsulated in the nanosystem for the preparation of various nanocomposites (Fitzpatrick, L.R. and T. Woldemariam "Small-molecule drugs for the treatment of inflammatory bowel disease" (2017): 495-510).

[0029] Tofacitinib was filled in NE at a concentration (10 mg / kg) required for therapeutic activity in mice. Formulation optimization also considered the stability of the system with respect to encapsulation efficiency, size, and surface charge (all important parameters for stable and controlled drug release). The optimized formulation reached a drug loading (DL%) of 2.2%.

[0030] The size distribution and surface potential of NE droplets were determined using a Malvern Zetasizer (registered trademark) Nano ZS instrument (Malvern Instruments S.A., Worcestershire, UK). The particle size was measured by dynamic light scattering (DLS) at a scattering angle of 173° at 25 °C. The ζ potential was calculated from the average electrophoretic mobility measured for samples diluted in milliQ water. The stability of the blank and TFC-NE in the colloidal suspension was followed for 6 months when stored at 4 °C. At the planned time points, the particle size, polydispersity index (PDI), and ζ potential were measured.

[0031] To quantify the TFC filled in the nanosystem, NE was dissolved in MeOH to disrupt the particle structure and analyzed by RP-HPLC. The liquid chromatography system consisted of a UHPLC Aquity Arc equipped with a diode array detector (PDA), a binary pump, and a septum injection valve with a fixed 10 μL loop. The analyte was monitored at 254 nm. Chromatographic analysis was performed on a Kinetex C18 column (Phenomenex, Torrance, CA, USA) with a size of 150 mm × 4.6 mm and a particle size of 5 μm. A mixture of methanol and water with a 50:50 v / v ratio was used to elute the compounds, and the column temperature was set at 30 °C [VK, Dhiman V, Giri KK, Sharma K, Zainuddin M, Mullangi R., Development and validation of a RP-HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study, Biomed Chromatogr. September 2015;29(9):1325~9, doi:10.1002 / bmc.3426.Epub 2015 Jan 26, PSID:25622797]. The HPLC calibration curve was linear in the concentration range of 20 μg / mL to 100 μg / mL (R 2 = 0.99). The method was verified according to the ICH Q2(R1) guidelines. The limits of detection and quantification (LOD and LOQ) were 6.17 μg / mL and 18.69 μg / mL, respectively. Diluted samples were filtered using a 0.22 μm nylon filter (Whatman GmbH, Dassel, Germany) before injection into the HPLC system. The drug concentration was tracked for 6 months.

[0032] The nanocomposites were prepared by mixing two concentrations of PS with a predefined amount of NE (a constant NE concentration of 5% w / v) such that different behaviors regarding particle release were observed, resulting in final PS concentrations of 0.5 w / v and 2% w / v. The mixture of PS and NE was diluted with a saline solution at pH 1.8 if necessary and vortexed for 5 minutes to obtain a homogeneous final formulation. The two final nanocomposites are defined as NE_PM0.5 and NE_PM2. In various embodiments, the amount of the peptoproduct can vary as described at “peptide concentration,” but the concentration of NE can also vary, for example, from about 2% v / w to about 50% v / w, from about 2% v / w to about 40% v / w, from about 2% v / w to about 30% v / w, from about 3% v / w to about 30% v / w, from about 3% v / w to about 20% v / w, from about 3% v / w to about 20% v / w, from about 4% v / w to about 25% v / w, from about 4% to about 15%, from about 5% v / w to about 20% v / w, from about 4.5% to about 10.%, from about 5% to about 20%, from about 5% v / w to about 12% v / w (from about 5% to about to about 12% v / w), from about 5% v / w to about 10% v / w, and further, for example, at least 1% v / w, at least 2% v / w, at least 3% v / w, at least 4% v / w, at least 5% v / w, at least 7% v / w, at least 9%, at least 10% v / w, at least 12% v / w, at least 15% v / w, at least 20% or more.

[0033] Rheology measurements were performed using an MCR 302 rheometer (Anton Paar, Les Ulis, France) equipped with a 25 mm cone-plate geometry. The temperature was set at 25 °C. The applied strain (γ%) was fixed at 0.5% within the linear viscoelastic region based on previous amplitude sweep tests. The apparent storage modulus and loss modulus of the empty hydrogel and the nanocomposites were measured by a frequency sweep test over a frequency range of 100 rad / s to 0.0264 rad / s.

[0034] Rheology can be used to evaluate the average mesh size of hydrogels [Karvinen J, Ihalainen TO, Calejo MT, Joenkkaeri I, Kellomaeki M, Characterization of the microstructure of hydrazone crosslinked polysaccharide-based hydrogels through rheological and diffusion studies, Mater Sci Eng C Mater Biol Appl. January 1, 2019;94:1056~1066, doi:10.1016 / j.msec.2018.10.048. Epub 2018 Oct 17, PSID:30423686]. The average mesh size (ξ, nm), which is defined as the distance (Å) between crosslinking points, can be calculated from Equation 5: [Equation] where in this equation, G’ is the storage modulus, NA is Avogadro's constant (6.022 * × 23 ), R is the gas constant (8.314 J / Kmol), and T is the temperature (310 K).

[0035] The crosslink density of the hydrogel can also be evaluated. The crosslink density (n e , mol / m 3 ), which describes the number of elastically active junctions in the network per unit volume, can be calculated from Equation 6: [Equation] as follows.

[0036] The inventors analyzed data obtained by frequency sweep analysis using a cone / plate geometry for empty hydrogels at two different concentrations of PS, 0.5% w / vol and 2.0% w / vol, and individual nanocomposites included at 5% w / vol (see above).

[0037] (Self - organizing peptides for use with nanocomposites) The rheological properties of the self - organizing peptides PURASTAT (RADA16; (SEQ ID NO: 1)), IEIK13 (SEQ ID NO: 2), QLEL12 (SEQ ID NO: 3), and KLD12 (SEQ ID NO: 4) (each having unique physical and biochemical properties) are suitable for the present invention and have been previously disclosed. PURASTAT is the synthetic peptide Ac - RADARADARADARADA - CONH 2 (SEQ ID NO: 1) and potentially its truncated fragments, and is commercially supplied as a 2.5% wt / vol solution in water (3 - D Matrix, Ltd., Japan). Its SAP, IEIK13 (SEQ ID NO: 2), exhibits different gelation characteristics when applied as a solution in vitro and when contacted with biological fluids (e.g., blood) in vivo or under in - vivo - like conditions. Both of these SAPs form nanofiber matrices and viscous hydrogels at a certain range of concentrations at approximately neutral pH. The SAPs disclosed in the present application share this and other characteristics despite having various compositions.

[0038] PURASTAT is composed of the amphiphilic self - organizing peptide RADA - 16 (aspartic acid, arginine, alanine; (SEQ ID NO: 1)) supplied in a sterile state at 2.5% (wt / vol) in water (3 - D Matrix). Its solution has a pI of 7.2, a pK1 of 1.79, and a pK2 of 12.58, and exhibits various behaviors and properties at various pH values. At pH 2.2, PS is a viscous solution. Once the gel is contacted / ruptured, reorganization is slow due to strong repulsive (+) electrostatic interactions. At pH 2.5 - 4, PS forms a semi - rigid viscous solution. Gel formation is induced by hydrophobic and charge - charge interactions. Once the gel is ruptured, reorganization is fast due to weak electrostatic interactions. Between pH 4 and pH 7.5, the self - organizing peptide forms a rigid hydrogel. Nanofiber formation is due to hydrophobic and attractive charge - charge interactions.

[0039] Both RADA16 (SEQ ID NO: 1) and IEIK13 (SEQ ID NO: 2) have been shown to be useful in promoting rapid hemostasis when applied to leaking biological tissue, and when forming a hydrogel matrix integral with the wound tissue, they promote normal healing over time without scarring or lack of healing. As reported by Katsuyama et al. (Minimally Invasive Therapy & Allied Technologies, 29(5):283-292 (2020)), IEIK13 (referred to as TDM-623 in that reference) forms a stiffer gel (i.e., having a higher storage modulus G’) when exposed to physiological conditions compared to RADA16 (referred to as TDM-621 in that reference), and this correlated with improved hemostasis when the product was applied to punch hole injuries in porcine livers. Although statistically significant improvements in hemostasis compared to RADA16 have been reported, these authors also reported that there was no infiltration of inflammatory cells due to the presence of RADA16 gel or IEIK13 gel after application of SAP solution to individual wounds in short-term studies. In a more recent study, IEIK13 ((SEQ ID NO: 2); (referred to as TDM-623 in that reference)) was tested as a hemostatic agent administered endoscopically to leaking wounds created in the walls of porcine stomachs and / or duodenums and appeared to be successful, but no control group was included (Kubo et al., Endoscopic application of novel, infection-free, advanced hemostatic material: Its usefulness to upper gastrointestinal oozing (2021); doi.org / 10.1002 / deo2.25). As also found in the studies cited above, hemostasis was achieved in both heparin-treated and non-heparin-treated animals. Given the acidic pH in the stomach, it should be noted that application of IEIK13 still resulted in hemostasis of gastric bleeding.

[0040] The SAP used in the present invention is completely synthetic and has no risk of infection from animal-derived products. Furthermore, it has been reported in a number of studies that RADA16 and IEIK13 do not themselves promote either immune response or inflammation (for example, Katsuyama et al.; Kubo et al.).

[0041] Other disclosures detail the rheological and gelation characteristics of RADA16 (SEQ ID NO: 1) and other SAPs aimed at directly applying an SAP solution to tissues (see, for example, U.S. Patent No. 10,654,893). In the present disclosure, PURASTAT (RADA16 (SEQ ID NO: 1) supplied at 2.5% wt / vol in water) is embedded in nanoemulsion (NE) particles having a hydrophobic core that carries either a fluorescent label or one of two hydrophobic drugs, tofacitinib (TFC) or curcumin (CCM), that have already been used to treat inflammatory bowel disease (IBD), creating a nanocomposite shown herein to have improved therapeutic efficacy when delivered by forced oral administration in a mouse model of colitis compared to either PURASTAT alone or drug-loaded NE particles alone. In a preferred embodiment, the peptide concentration prior to formation of the nanocomposite in the present invention is preferably within a range of about 0.05% to about 4% in solution, or any other concentration listed in "Peptide Concentration" below. Further, it is shown that the dry nanocomposite gel rapidly rehydrates upon contact with water while simultaneously maintaining the NE intact within the nanofiber matrix. Oral administration of the dry nanocomposite encapsulated within a pH-sensitive capsule, sustained-release capsule, or other capsule has the ability to provide delivery of the nanocomposite to one or more regions of the gastrointestinal tract (GIT), in which regions the capsule dissolves to release the nanocomposite such that the nanocomposite associates with and rehydrates in the GIT or with damaged or diseased tissue and releases an effective amount of the drug(s) carried by the NE to treat the target region. As described, the SAP hydrogel component of the nanocomposite system aids in the healing of damaged or diseased tissue while simultaneously surrounding the drug-loaded NE, thereby providing local sustained-release of bioactive drugs that could otherwise be degraded before reaching their target by other means.

[0042] The use of D - amino acid - containing SAPs (including RADA16 and IEIK13) as a hydrogel component of a nanocomposite system is included in embodiments of the present invention, particularly when a slower rate of in vivo degradation and / or absorption may be desirable. Appropriate peptide concentrations for RADA16, IEIK13, and all other peptides are listed in "Peptide Concentrations" below.

[0043] In some embodiments of the present invention, the SAP is KLD12 (SEQ ID NO: 4). In some specific embodiments, the SAP is IEIK13 (SEQ ID NO: 2), and the peptide concentration prior to the formation of the nanocomposite in the present invention is preferably within a range of about 0.05% to about 2.0% in water, considering that if the concentration exceeds this in water, only this peptide may be too viscous to form a useful hydrogel for the nanocomposite (see US Patent No. 10,654,893, Table 1 therein). However, when other components (e.g., NE) are added to the IEIK13 (SEQ ID NO: 2) solution and / or bound to the peptide (e.g., a physiologically active peptide and / or a medicine), the rheological properties of the solution can change. However, as disclosed in the following studies, PURASTAT (SEQ ID NO: 1) did not show a change in rheological properties when filled with up to 10% wt / vol of NE.

[0044] (Peptide Concentration) - The rheological properties of the peptide compositions described previously (U.S. Patent No. 10,654,893) can be controlled by the selection of the peptide concentration, for example, through the selection and / or adjustment of the peptide concentration, if particularly preferred for a particular adaptation or use of the composition. For a number of SAPs, it has been shown in vitro that the hardness of the composition increases substantially linearly with the peptide concentration. Any of the peptides described in Table 2 can be used at a concentration in a non-swelling solution in the range of from about 0.05% to about 5%, from about 0.05% to about 4%, from about 0.5% to about 4%, from about 0.5% to about 3.5%, from about 0.5% to about 3%, from about 0.5% to about 2.5%, from about 0.5% to about 2%, from about 0.5% to about 1.5% or at about 1%, about 2%, about 2.5%, about 3%, about 4%, about 5% (all v / w).

[0045] In vitro, the rheological properties achieved at a specific peptide concentration vary depending on the entity of the peptide. For example, the storage modulus G’ of 1.5% KLD12 (SEQ ID NO: 4) in water was found to be approximately 350 Pa, similar to the storage modulus G’ of 2.5% RADA16 (SEQ ID NO: 1) in water under the same test conditions. The storage modulus G’ of 1% IEIK13 (SEQ ID NO: 2) in water (about 700 Pa) was found to be similar to the storage modulus G’ of 2.5% KLD12 (SEQ ID NO: 4) in water under the same test conditions and higher than the storage modulus G’ of 2.5% RADA16 (SEQ ID NO: 1) in water (about 350 Pa) (U.S. Patent No. 10,654,893 - Tables 3 and 3A). Overall, the order of rheological strength among these compositions is IEIK13 (SEQ ID NO: 2) > KLD12 (SEQ ID NO: 4) > RADA16 (SEQ ID NO: 1), and thus, when the peptide concentration in water was the same in each case, the composition of IEIK13 (SEQ ID NO: 2) exhibited a greater rheological strength than that shown by the composition of KLD12 (SEQ ID NO: 4), and then, this composition of KLD12 (SEQ ID NO: 4) exhibited a greater rheological strength than that shown by the composition of RADA16 (SEQ ID NO: 1). Considering the new results obtained by filling nanoemulsion particles (NE) into RADA16, the previous characterization of various SAPs provides a reasonable prediction that the formulations of nanocomposites containing various SAPs embedded with drug-loaded NE also exhibit rheological properties similar to or the same as those of simple SAPs.

[0046] Thus, in some embodiments of the present invention, those SAPs are of formulas I-IV: ((Xaa neu -Xaa + ) x (Xaa neu -Xaa - ) y ) n (I) ((Xaa neu -Xaa - ) x (Xaa neu -Xaa + ) y )n (II) ((Xaa + -Xaa neu ) x (Xaa - -Xaa neu ) y ) n (III) ((Xaa - -Xaa neu ) x (Xaa + -Xaa neu ) y ) n (IV) contains an amino acid residue sequence according to one or more of the following, where Xaa neu represents an amino acid residue with a neutral charge; Xaa + represents an amino acid residue with a positive charge; Xaa - represents an amino acid residue with a negative charge; x and y are integers independently having values of 1, 2, 3, or 4; n is an integer having a value of 1 to 5.

[0047] The above and other peptides suitable for the method of the present invention are similar to RADA16, and they include any of the following listed in Table 2 as well as modified peptides and peptidomimetics further listed below (all with appropriate N-terminal carboxylation and C-terminal amidation of the peptide), and appropriate concentrations listed in the above "peptide concentration".

Table 2

[0048] In some embodiments of the present invention, the peptoproduct further includes an amino acid sequence that interacts with the extracellular matrix, and the amino acid sequence immobilizes SAP to the extracellular matrix (e.g., RGD).

[0049] In other embodiments, the amino acid residues in the SAP can be naturally occurring amino acid residues (which may be synthetic or not of animal origin) or amino acid residues not naturally occurring. Naturally occurring amino acids can include amino acid residues encoded by the standard genetic code, while amino acids not naturally occurring can include non-standard amino acids (e.g., amino acids having a D-configuration instead of an L-configuration, or combinations of D- and L-amino acids), as well as amino acids that can be formed by modification of standard amino acids (e.g., pyrrolidine or selenocysteine). Suitable non-naturally occurring amino acids include, but are not limited to, D-alloisoleucine (2R,3S)-2-amino-3-methylpentanoic acid, L-cyclopentylglycine (S)-2-amino-2-cyclopentylacetic acid. In some embodiments, the SAP used in the present invention contains only naturally occurring amino acids, or only non-naturally occurring amino acids (e.g., D-amino acids) (e.g., RADA16 composed of D-amino acids or IEIK13 composed of D-amino acids); or a combination of D- and L-amino acids. RADA16 composed of D-amino acids, as well as other SAPs, can be used in the methods of the present invention to potentially reduce the in vivo degradation of the hydrogel matrix, thereby increasing the residence time of the hydrogel at the site of attachment, which in turn can affect the retention of solutes (e.g., drugs) within the matrix, the ingrowth of tissue into the matrix, and tissue healing.

[0050] In other embodiments, another type of material that can self-organize and mimic its SAP is a peptidomimetic. As used herein, a peptidomimetic refers to a molecule that mimics a peptide structure. Peptidomimetics have general characteristics (e.g., amphiphilicity) similar to those of their parent structure, the polypeptide. Examples of such peptidomimetic materials are described in Moore et al., Chem. Rev. 101(12), 3893-4012 (2001). The peptidomimetic materials used in the present invention can be classified into the following four categories: α-peptides, β-peptides, γ-peptides, and δ-peptides. Copolymers of these peptides can also be used. Examples of α-peptide peptidomimetics include, but are not limited to, N,N'-linked oligoureas, oligopyrrolinones, oxazolidin-2-ones, azatides, and azapeptides. Examples of β-peptides include, but are not limited to, β-peptide foldamers, α-aminooxy acids, sulfur-containing β-peptide analogs, and hydrazinopep tides. Examples of γ-peptides include, but are not limited to, γ-peptide foldamers, oligoureas, oligocarbamates, and phosphodiesters. Examples of δ-peptides include, but are not limited to, alkene-based δ-amino acids and carbo peptoids (e.g., pyranose-based carbo peptoids and furanose-based carbo peptoids).

[0051] In certain embodiments, the SAP is AC5®, AC5-V®, AC5-G™ or TK45 (also known as AC1), made and available from Arch Therapeutics, Inc. (see www.archtherapeutics.com). Each of these self-assembling peptides and other self-assembling peptides disclosed herein are capable of forming hydrogels when applied to living tissue (e.g., in situ) at near neutral pH. Generally, the SAP concentration in water ranges from about 1 weight / volume % to about 5 weight / volume %, but this range is not exclusive. For example, the aqueous concentration at which the IEIK13 (SEQ ID NO:2) peptide forms a hydrogel matrix when exposed to physiological conditions is generally between about 0.5% and about 2.5%. However, the self-assembly (or lack thereof) of this and other peptides in an aqueous environment depends on a number of variables, including pH, ionic concentration, concentration and composition of the SAP itself, type of ions present, and many other factors. Data characterizing and comparing the properties of RADA16 (SEQ ID NO:1), IEIK13 (SEQ ID NO:2) and KLDL (SEQ ID NO:4) under various conditions can be found in U.S. Patent No. 10,654,893, the entire contents of which are incorporated herein by reference.

Example

[0052] (Example) (Example 1. Rheological Characterization) Frequency sweep measurements were performed at variable frequencies with the strain amplitude in the linear viscoelastic region (LVR) determined by amplitude sweep measurements. As shown for Figure 2A, for all hydrogel composites, G’ was higher than G’’ (elastic response was stronger than viscous response) and independent of frequency. G’ was also parallel to G’’. The ratio of G’’ to G’ (tanδ) was <0.2 for all hydrogels, and thus their structures were considered strong. Due to these findings, these hydrogels were considered to be stable and strongly cross-linked gels. The G’, G’’, and G of the hydrogels shown in Table 2 * were determined at 1.27 rad / s. Their results showed that the G’ (and G * ) of those hydrogels were lower at the minimum concentration. Table 1 above shows a comparison of the NE used in this example.

Table 3

[0053] The microstructure plays an important role in controlling hydrogel properties. It is also an important factor when cells or drugs are encapsulated inside the hydrogel. The microstructure of PURASTAT and the nanocomposite was evaluated by using a rheology-based method. The average mesh size (ξ) of the hydrogel was calculated using Equation 5. The ξ of the empty hydrogels was 144.65 ± 1.03 nm for PS0.5 and 40.63 ± 2.86 nm for PS2, respectively, while the ξ of the hydrogels embedded with NE was 127.39 ± 3.60 nm for NE_PM0.5 and 35.71 ± 0.87 nm for NE_PM2, respectively. These results are shown in Figure 2B. The ξ of the hydrogel decreased when the polymer concentration increased, while the presence of NE showed no effect on the rheological structure of the hydrogel. The cross-linking density (n e ) of the hydrogel was calculated using Equation 6. The calculated parameters are shown in Table 2 and Figure 2C. n eThey were 0.55 ± 0.01 mmol / m for PS 0.5% respectively 3 and 24.76 ± 4.82 mmol / m for PS 2% 3 . The nanocomposites showed similar results of 0.80 ± 0.07 for NE_PM0.5 and 36.46 ± 2.59 for NE_PM 2 respectively. The n of the hydrogel e decreased when the concentration of the hydrogel decreased.

[0054] (Example 2. Drying of Nanocomposites) The system was converted to dry solid powder using lyophilization technology. Based on the slightly increased size of NE after filling in the PS hydrogel compared to the size measured before filling, PURASTAT seemed to generate a shell surrounding NE. Also, no additional cryoprotective substance was required. The resulting cake was white, smooth and dense. Resuspension in water was easy and gelation was immediate. NE maintained their properties and there was no significant change in size. The higher PDI values could be attributed to the presence of PS in the solution (Table 4).

Table 4

[0055] For samples with NE percentages of 0.5 and 1, the dried nanocomposites appeared as white, dense cakes. Samples with 2.5% NE and samples with 5% NE became white, brittle and dense cakes when dried. All of these samples rehydrated easily and reformed hydrogels immediately when water was added and gently shaken by hand. In comparison, samples with 12.5% NE formed white, dense cakes upon lyophilization but did not rehydrate.

[0056] (Example 3. Circular Dichroism Characterization of Nanocomposite Formulations for In Vivo Studies) Using CD experiments, we understood whether the incorporation of nanoparticles has a detrimental effect on the initial self-organization of peptides through analyzing the β-sheet peak. This β-sheet peak is an indicator of nanofiber formation (Lu, L. and Unsworth, L. D. PH-Triggered Release of Hydrophobic Molecules from Self-Assembling Hybrid Nanoscaffolds, Biomacromolecules, 17, 1425~1436 (2016)). Control experiments were carried out using CD characterization of nanoparticles without peptides, in which no response was observed as a function of wavelength. The peptide control showed a typical β-sheet structure for a system without nanoparticles (minimum at 217 nm~218 nm, maximum at 195 nm~206 nm). The content of this secondary structure was further quantified using CDNN software to obtain the percentages shown in Figure 3. As can be seen in the drawing, no obvious difference regarding the secondary structure was detected when PS was incubated with NE.

[0057] (Example 4. Model Drug Release Study) After the characterization of the nanocomposite, the ability of the drug-loaded NE to be released from the hydrogel was investigated. Curcumin is an active substance mainly characterized by low water solubility, poor stability in body fluids, high metabolic rate, rapid clearance, reduced absorption in the gastrointestinal tract (GIT) and limited bioavailability. These are common features among many low-molecular-weight drugs commonly used for the treatment of many diseases (Yavarpour-Bali et al., Curcumin-loaded nanoparticles: A novel therapeutic strategy in treatment of central nervous system disorders, International Journal of Nanomedicine, Vol. 14, 4449 - 4460 (2019)). Curcumin (CCM) was loaded into the nanoemulsion to track its release from the nanoparticles in the PS nanocomposite. The release of NE from NE_PM0.5 and NE_PM2 was tested in phosphate buffer at pH 4 and 7.4, respectively. 1 g of the nanocomposite in hydrogel form was weighed in a vial and 4 mL of the individual buffer was added on top. At the planned time points, the supernatant was removed and replaced with the same volume of fresh buffer. The amount of curcumin released (proportional to the amount of NE released) was quantified by RP-HPLC.

[0058] The cumulative results of these studies are shown in Fig. 4. The results of this model study highlight the effects of both peptide concentration and pH on drug release. The higher the PS concentration, the slower its release. These results are consistent with the rheology study of this model study. The rheology study of this model study indicates that in the presence of a larger mesh and lower crosslink density for PS0.5, NE can be released faster in PS2 (which is characterized by a higher crosslink density and smaller mesh size) where they are filled in CCM than in PS2. On the other hand, pH also affects hydrogel behavior. The isoelectric point of the RADA16 (SEQ ID NO: 1) peptide is 7.2, which means that at a lower pH, the mesh and structure of the hydrogel are complete, while a shift to a higher pH brings about a change in its drug release characteristics.

[0059] (Example 5. In Vivo Model of Colitis) In the first stage of these in vivo studies, we evaluated experimentally determined data related to DSS-induced colitis in mice, making the induced colitis in mice not as extreme as to be incurable. As shown in Fig. 6, the mice were divided into four groups. In G1 and G2, the mice were given 3% DSS in drinking water for 4 days. Subsequently, the G1 mice and G2 mice were sacrificed on the 7th and 14th days, respectively. The G3 mice and G4 mice were given 3% DSS in drinking water for 7 days, and then the G3 mice and G4 mice were sacrificed on the 7th and 14th days, respectively. From the start to the sacrifice of this study, all mice were scored based on the following factors: [Table 5]

[0060] The DAI scores observed experimentally for those four mouse groups are plotted in Figure 5. These results showed that administration of 3% v / v DSS in drinking water for 7 days resulted in extremely severe colitis induction, while administration for 4 days led to a slow and complete recovery of those animals. In fact, in these studies, it was important not to cause an extreme condition that would be too difficult to treat. For this reason, the incubation time with DSS for 4 days was selected for the next study, followed by treatment / recovery until day 10, which showed good progress towards recovery in terms of gross and histological evaluations at stage 1.

[0061] (Example 6. In vivo distribution of PS_NE filled with fluorescent dye in the digestive tract of mice with DSS-induced colitis) In this example, 3% DSS in drinking water was given to IBD mice for 4 days, while normal water was given to control healthy mice. At the end of the 4th day, mice in both groups were given, by forced oral administration, NE alone (NE) filled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD), or a nanocomposite containing its DiD-NE (DiD-NEPM). This mimicked the first treatment given to those mice in the efficacy study that was planned to be described. Those mice were sacrificed at the planned time points of 1 hour, 3 hours, 6 hours, and 24 hours, and their digestive tracts were taken for further study of the fluorescence distribution in the GIT. The last time point at 24 hours (not shown) showed no fluorescence. Fluorescence images of the tissues taken at earlier time points are shown in Figure 6. A semi-quantitative analysis of the images shown in Figure 6 using the software Wasabi is shown in Figure 7. Furthermore, qualitative fluorescence obtained by LSFM (light sheet fluorescence microscopy) after tissue clearing was observed in tissues derived from 2 of the IBD mice, one given NE and the other given NEPM. Both were sacrificed 3 hours after forced oral administration.

[0062] The images shown in Figure 8 are particularly prominent. For these images, the upper colon of IBD mice that received NE-DiD (A) or PS-NE-DiD (B) in solution by gavage was collected, fixed in 4% v / v PFA for 24 hours, and then transferred to PBS at pH 7.4 for qualitative visualization of the preferential accumulation of the dye in the tissue. The tissues were cleared using the X-clarity tissue clearing system, and the fluorescence of those particles in the ex vivo colon tissue was observed using light sheet fluorescence microscopy. Figure 8A shows little fluorescence, while Figure 8B (derived from IBD mice treated with PS filled with DiD-NE) shows fluorescence from DiD distributed throughout most of the tissue sample. This finding indicates that the inflamed colon tissue takes up the nanocomposite NE particles very well, while the tissue uptake of NE-DiD particles is very limited. Only two mice were compared in this study, but again, the results are prominent.

[0063] (Example 7. Efficacy of drug-loaded nanocomposite systems in treating DSS-induced colitis) The effectiveness of the drug-loaded nanocomposites compared with various controls was tested using mice of the same strain as those used in Example 5. Based on the results from the previously described Stage 1 in vivo study, all mice (except the G1 healthy control group) were given 3% v / v DSS in drinking water for 4 days. After 4 days, all mice were given normal drinking water (i.e., without DSS). After the 4th day, the DSS-treated mice were orally administered various components of the nanocomposite containing NE loaded with tofacitinib (TFC) as shown in Table 4 by forced oral administration on the 5th, 7th, and 9th days. The condition of the mice was observed as in Example 5 / Stage 1 and scored according to the criteria in Table 5. Figure 9A shows the percentage of body weight loss of mice in each group over time, and Figure 9B plots the DAI scores. Figure 10 (derived from Figure 9B) more clearly shows that statistically significant improvements were observed on the 7th and 8th days in the DAI scores for the IBD mice administered PSNE containing TFC compared to the other IBD mouse groups. A group of mice in each group was sacrificed on the 5th, 7th, and 10th days, and then tissues were collected for further analysis.

[0064] In Figure 9A, the percentage of body weight loss can be observed for all groups involved in this study. G1 showed no decrease in body weight and increased slightly over time. All other groups showed body weight loss starting from the 5th day, when the effect of DSS began to be visually shown. According to the DAI data, G6 was the only group that showed good results compared to other groups even regarding only body weight loss. In Figure 9B, the DAI scores obtained for all groups involved in this study can be observed. While G1 showed a low score, all other groups followed a similar behavioral profile, peaked around the 7th day, and then continued with a slow remission. The only group that showed a better profile was G5, where a slight decrease in its DAI score was detected after the administration of the first treatment on the 5th day. This could be due not only to a slower long-term release of the drug from the nanosystem but also mainly to a slower long-term release of the nanosystem from the hydrogel. Table 6: Stage 2 In Vivo Study of the Efficacy of NE-PM Nanocomposites Filled with Tofacitinib Group 1 (G1): Healthy mice (n = 5), negative control Group 2 (G2): DSS-induced colitis mice (n = 15), positive control Group 3 (G3): DSS-induced colitis mice (n = 10), forced oral administration of TFC Group 4 (G4): DSS-induced colitis mice (n = 10), forced oral administration of NE filled with TFC (TFC_NE) Group 5 (G5): DSS-induced colitis mice (n = 10), forced oral administration of NE filled with TFC embedded in PS (PM_NE_TFC) Group 6 (G6): DSS-induced colitis mice (n = 10), forced oral administration of empty PS Group 7 (G7): DSS-induced colitis mice (n = 10), forced oral administration of empty NE.

[0065] As shown in Figure 11, macroscopic analysis (used in the prior art) was performed by dividing the weight (g) of each colon collected from each mouse by its length (cm) shown, for example, in Figure 11B. This data (plotted in Figure 11A) shows a statistically significant improvement (i.e., lower g / cm) for G3 (treated with simple TFC) and G5 (PS_NE_TFC mice) compared to untreated IBD mice in G2.

[0066] Figure 12 shows the results from the quantification of myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration in the colonic mucosa. The method described here was used: www.sigmaaldrich.com / FR / fr / product / sigma / mak068?gclid=Cj0KCQiA15yNBhDTARIsAGnwe0U8JudlWDlwyCYqrvusn2PQB3WnPk-ec3mxqu_5eAp6vwAYiItUdmYaAtC3EALw_wcB. These results indicate that the MPO activity was lower in colonic tissue derived from IBD mice treated with PS_NE_TFC, particularly when compared to G3 mice treated with simple TFC.

[0067] Exemplary histological results of colon tissues stained with HPS are shown in FIGS. 13 to 18 for mice from various groups as indicated.

[0068] The macroscopic results from this study are plotted in FIG. 9. As shown more clearly in FIG. 10, mice treated with the nanocomposite filled with TFC showed a statistically significant improvement in the DAI scores on days 7 and 8 of this study compared to other treatment groups.

[0069] (Example 8. Comparison of in vivo distribution in healthy mice and IBD-induced mice) To evaluate the ability of NE to retain in the inflamed GIT and healthy GIT or the ability of the hybrid system PS-NE to retain, fluorescent dye DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate) was loaded into the nanoparticles and analyzed by near-infrared fluorescence imaging for forced oral administration. After 4 days of colitis induction using 3% w / v DSS (as defined in previous studies), observations were made at 1 hour, 3 hours, and 6 hours on day 5. Time points were selected according to previous studies (Rosso A., Andretto V. et al., Nanocomposite sponges for enhancing intestinal residence time following oral administration, J. Control. Release (2021) 333:579-592), considering that mice have a total GI transit time of about 6 hours and that most of the intestinal contents are located in the small intestine and cecum after 3 hours. To perform a semi-quantitative analysis of the fluorescent dye distribution, organs were collected and ex vivo images were taken as shown, for example, in FIG. 8. As shown in FIG. 9, images collected at various time points were processed to extract various information regarding the residence time, targeting ability, and potential toxic effects of the formulation.

[0070] As shown and analyzed in FIG. 6, the in vivo distribution of NE in healthy animals shows a progressive passage from the stomach to the rest of the intestinal region, with relative fluorescence in the colon at 3 and 6 hours, which is indicative of a progressive excretion of a detectable amount of fluorophore. In the case of the in vivo distribution of NE in IBD mice, the overall excretion from the stomach is faster, and the amount of remaining DiD in the GIT at 6 hours is concentrated in the colon.

[0071] Regarding the administration of the hybrid composite, as shown in FIG. 7B, the inventors observed a similar trend showing fluorophore accumulation in the stomach over time in both healthy and IBD mice. The main difference between these two groups of animals is the preferential accumulation of detectable fluorescence in the small intestine for IBD mice, while for healthy mice, the passage through the small intestine appears to be faster, as the inventors detected stronger fluorescence intensity in the colon.

[0072] (Example 9. Functional effect of self - assembling peptide alone) The group G6 samples described in Example 6 (DSS - induced colitis mice (n = 10), forced oral administration of self - assembling peptide alone) were further analyzed. FIG. 19A shows the mean of the ratio of colon weight / colon length per group of mice (n = 5). Similar to the results obtained for weight loss, group G6 showed a significantly better profile, in which the decrease in its DAI score was detected from the administration of the first treatment and reached its maximum effect at the peak of the profile (days 7 and 8). The MPO enzyme activity, as an indicator of granulocyte infiltration, was measured. The results are shown in FIG. 19B. The nanocomposite showed a significantly decreased activity of MPO (a decrease maintained when animals were treated with PS alone), while treatment with the free drug showed high intra - individual variability even when slight improvement was obtained.

[0073] Treatment with TFC alone (G3) resulted in a significant remission of the epithelial and muscular layer structures and a decrease in lymphocyte infiltration, which was further accentuated when the mice received the treatment double-encapsulated in the nanocomposite structure (G5). The group treated with hydrogel alone (G6) did not show a significant decrease in CD45+ cell infiltration as shown in Figure 19C, even when significant improvement was observed especially in its structural recovery with respect to G2.

[0074] When compared with free TFC treatment (G3), administration of PM-NE-TFC (G5) greatly decreased both the activity and density of neutrophils, resulting in a decrease in the myeloid cell population. The decreased effect on macrophages may be the result of changes in the relative percentages of various cell populations due to the strong decrease in granulocytes also shown by MPO analysis.

[0075] The total amount of T cells (CD3 + ) was strongly decreased by the presence of the drug, which was maintained when PM alone was administered. Nevertheless, when analyzed as the relative amount of total immune cells (CD45 + cells), the inventors did not observe any significant change in the frequency of either CD8 T cells or CD4 T cells, except for an increase in CD4+ T cells in the LP of animals administered PM only. Both TFC treatment and PM treatment significantly decreased the ratios of both the Th1 pro-inflammatory population and the Th17 pro-inflammatory population, but did not affect the Th2 population or the Treg population. PM alone showed a similar ability as both free and encapsulated forms of TFC in decreasing the ratios of both the Th1 pro-inflammatory population and the Th17 pro-inflammatory population.

[0076] Although these phenomena require further investigation, since T cell involvement is not required for the initial onset of DSS-induced colitis and indicates the result of major inflammation, the role of PM in reducing inflammatory cytokine production can interfere with the recruitment and differentiation of Th1 and Th17 populations, and as a result, it may reduce or even prevent the exacerbation of the innate immune response caused by Th1 cytokines and IL17A, the major product of the Th17 population.

[0077] Furthermore, the efficacy of TFC is preserved when administered in nanocomposite form. Not surprisingly, PM has shown interesting anti-inflammatory properties that can be utilized to improve the therapeutic efficacy of pharmacological treatments as well as to favor the mucosal healing process.

[0078] The interest in this developed nanocomposite made with PM and NE-TFC relies on the synergistic effect resulting from the co-administration of the molecule of interest and the hydrogel with intrinsic anti-inflammatory properties in a single dose.

Claims

1. A composition for oral administration for the treatment of a target gastrointestinal disease or gastrointestinal injury, Nanoemulsion particles (NE) comprising a hydrophobic liquid core and at least one hydrophobic surfactant (S1) and at least one hydrophilic surfactant (S2) in an outer layer, wherein the surfactant is a nonionic polar surfactant surrounding the core, and the core contains one or more therapeutically active agents; A composition comprising, wherein the NE is combined with a self-assembling peptide, the peptide, which is optionally in a dry form, organizes into a hydrogel matrix surrounding the NE when hydrated at a slightly acidic or near-neutral pH, thereby adhering to the intestinal wall and / or delivering the active drug to the intestinal wall when administered orally to a subject.

2. The composition according to claim 1, characterized in that it is administered in the form of a capsule or a tablet.

3. The composition according to claim 2, wherein the capsule or tablet dissolves slowly in the stomach or post-stomach intestine of the subject, thereby forming a depot in the digestive tract of the subject, and thereby releasing the composition into the intestine of the subject.

4. The composition according to claim 1, comprising surfactant S1 and surfactant S2.

5. The composition according to claim 4, wherein the surfactant comprises S1, which is polyoxyethylene (40) stealth (Myrj® 52), and S2, which is oleoyl polyoxy-6 glyceride (Labrafil® M1944CS).

6. The composition according to claim 4, wherein the ratio of S1 to S2 is between 1 and 5 in terms of weight / weight.

7. The composition according to claim 1, wherein the self-assembling peptide is one of the peptides selected from Table 1 (SEQ ID NOs: 1 to 24).

8. The composition according to claim 7, wherein the self-assembling peptide is provided in a dry form.

9. The composition according to claim 7, wherein the self-assembling peptide is RADA16 (SEQ ID NO: 1).

10. The composition according to claim 7, wherein the self-assembling peptide is IEIK13 (SEQ ID NO: 2).

11. The composition according to claim 1, wherein the active agent is selected from the group consisting of tofacitinib, curcumin, and budesonide.

12. A composition according to any one of claims 1 to 11 for treating a digestive disease or digestive injury, characterized in that it is administered orally to a subject requiring treatment for a digestive disease or digestive injury.

13. The composition according to claim 12, wherein the disease to be treated is inflammatory bowel disease, Crohn's disease, ulcerative colitis, or a lesion resulting from surgery or cancer treatment.

14. A method for producing the composition according to any one of claims 1 to 11, a) A step of producing nanoemulsion particles (NE) comprising a hydrophobic liquid core and at least one hydrophobic surfactant (S1) and at least one hydrophilic surfactant (S2) in an outer layer, wherein the surfactant is a nonionic polar surfactant surrounding the core, and the core optionally contains a therapeutically active agent, thereby producing the composition obtained first; b) A step of combining the initially obtained composition with one or more self-assembling peptides present in a solution, wherein the peptides can be assembled into a hydrogel when hydrated at a near-neutral pH, thereby producing a composite material; c) a step of drying the composite material to produce a powder or "cake," if necessary; and d) If necessary, a step of formulating the material suitable for oral administration to the target, Methods that include...

15. The method according to claim 14, wherein the composite material is dried to produce a powder or a "cake".

16. The method according to claim 14, wherein the composite material is encapsulated in a capsule or formulated as a tablet, and when the capsule or tablet is placed in the digestive tract of a target, either one can provide a depot for the release of the composite material.

17. The composition according to claim 14, wherein the core contains a therapeutically active agent.

18. The composition according to claim 1, wherein the active agent is selected from the group consisting of S1P receptor modulators, other JAK inhibitors, CCR9 antagonists, α4 integrin antagonists, and immunomodulators.

19. The method according to claim 14, wherein the active agent is selected from the group consisting of S1P receptor modulators, other JAK inhibitors, CCR9 antagonists, α4 integrin antagonists, and immunomodulators.

Images