This invention relates to an oral drug delivery agent. More particularly, this invention relates to an oral drug delivery agent for protein therapeutics.

DESCRIPTION OF THE PRIOR ART

Protein therapeutics are increasingly becoming a critical part of pharmaceutical treatment for a growing number of diseases. Advancements in biotechnology and a better understanding of the pathophysiology of clinical conditions has led to the recognition of a growing number of proteins and peptides as potential therapeutics. There is currently a scarcity of non-invasive oral mode of administration for protein therapeutics. Proteins are naturally hydrophilic molecules with poor absorbance through the intestines and are prone to degradation by harsh pH and enzymatic action (Curto et. al. (2011)) ¹ .

Perhaps, the first ever protein molecule used as a therapeutic is insulin, the cornerstone of treatment for type 1 and type 2 diabetes mellitus. The only therapeutically deliverable form of insulin presently available is injectable insulin. Regular and very frequent subcutaneous administrations are associated with low patient compliance and multiple injection site injuries. It is not currently possible to orally administer insulin as insulin is a peptide that is known to be not resistant to stomach acid or enzymatic degradation. Oral drug administration is generally recognized as delivery of drugs through the gastrointestinal (GI) tract where the starting point for a drug is in the mouth or buccal cavity and absorption can take place at different portions of the small and large intestines (Zaman R. et. al. (2016)) ² . The GI tract comprises several organs with each organ having its own pH and enzymatic environment. The stomach and small intestines have pH environments and secretary enzymes that specialize in modifying and breaking peptide and protein molecules and these organs can destroy any protein drug that passes through them non-discriminatorily (Allen C. et. al (2011)) ³ . In addition to pH and enzymes, another problem to be overcome is intestinal absorption of high molecular weight protein molecules in intact form. The intestinal wall is covered with a mucus layer, which is a combination of glycoproteins and bicarbonate ions. Beneath the mucus is the epithelium, the basement membrane (non-cellular layer) and a layer of submucosa that holds blood vessels and lymphatic ducts. There are different modes of uptake of molecules (phagocytosis, absorption through Peyer's patches or endocytosis by enterocytes) depending on the size, surface charge, hydrophilic or hydrophobic nature of the molecule (Banga A. K. (2006)) ⁴ . Absorption of an intact protein generally fails due to its size and hydrophilic nature.

There have been a number of strategies proposed for an oral protein formulation with the most common approach being to attach a protein molecule to another molecule that envelops and creates a carrier for it. Nanoparticles are candidates for this.

US 8,859,004 B2 discloses a pH-sensitive insulin-loaded nanoparticle for oral insulin delivery. The nanoparticle comprises an enteric coating polymer (a pH-sensitive polymer), hydrophobic material, internal stabilizer, external stabilizer content and insulin. A modified double emulsion solvent evaporation method is used to prepare the nanoparticle.

US 9,101,547 B2 discloses an enteric-coated capsule containing insulin-loaded cationic nanoparticles for oral insulin delivery. The enteric-coated capsule encloses a plurality of nanoparticles and a solubilizer. Each of the nanoparticles comprises a polycationic polymer, a biodegradable polymer, insulin and a stabilizer.

Polymeric nanoparticles, such as poly(lactide-co-glycolide) (PLGA) and its derivatives have also been explored. Insulin incorporated into a blended polymer of polyfumaric anhydride (FA) and PLGA 50:50 (FA: PLGA) showed a reduction of glucose load in fasted rats for 3 hours (Mathiowitz E. et. al. (1997) ⁵ , Ensign L. M. et. al. (2012) ⁶ ) Another formulation of HP55-coated capsule containing PLGA/RS nanoparticle-loaded insulin gradually lowered glucose levels for 10 to 15 hours (Wu Z. M. et. al. (2012) ⁷ ). Another oral nano-insulin delivery tool under consideration is chitosan and its derivatives. US 9,828,445 B1 discloses modified chitosan particles for delivering oral insulin. Chitosan particles are amidated with an amino acid or a fatty acid and subsequently grafted with N-isopropylacrylamide and cross-linked to form modified chitosan particles. Insulin is then loaded onto the modified chitosan particles. pH- sensitive chitosan particles showed a 15-hour long effect of reducing blood glucose levels in diabetic rats, although, this started with a burst release (Pan Y. et. al. (2002) ⁸ ). Similarly, TBA (thiolated polymer 2-iminothiolane)-attached chitosan with incorporated insulin showed glucose-lowering effect for 24 hours in non-diabetic rats (Krauland A. H. et. al. (2004) ⁹ ).

However, due to the poor bioavailability of insulin following oral administration using such polymeric materials, the overall effect in lowering blood glucose level is undesirably limited.

Lysosome-entrapped insulin (LEI) are also being studied for oral administration of insulin (Molero J. E. et. al. (1982) ¹⁰ , Stefanov et. al. (1980) ¹¹ ), although, none of the formulations qualified for clinical trial. One example of this is oral insulin 338(1338), which was created by a combination of amino acid substitution and linkage to a carrier molecule. The clinical trial for this oral insulin candidate was discontinued as oral insulin 338(1338) was found to be only effective at a very high dose due to limited bioavailability, which would make production costs unacceptably high (Halberg I. et. al. (2018) ¹² ).

Another oral insulin formulation candidate is insulin tregopil (Biocon), a human insulin analogue attached to a methoxy-triethylene-glycol-propionyl moiety linked to the Lys-329 amino group and formulated with sodium caporate. In animal trials (dog), the bioavailability of this candidate was observed to be not very high (0.82% to 0.85%) (Drucker D. J. (2019) ¹³ , Gregory J. M. et. al. (2019) ¹⁴ ).

ORMD-0801 from Oramed is currently in Phase 2 clinical trial and is prepared by attaching insulin to a permeation enhancer, soybean trypsin inhibitor and a chelator. Pre-clinical stage bioavailability in animal studies was estimated at 5 to 8% (Arbit E. et. al. (2017) ¹⁵ ). Another oral insulin formulation under consideration is Diasome, a liver-targeting formulation. Current available data shows that oral dosing of Diasome is less effective compared to subcutaneous administration (Geho W. B. et. al. (2014) ¹⁶ ). This is likely due to Diasome's lack of ability to overcome the multiple barriers of the gastrointestinal tract before being absorbed across the intestinal epithelium.

Precipitation -based salt particles have also been considered for insulin loading for oral delivery. One such example is strontium-substituted carbonate apatite which has shown only very limited success in terms of binding and releasing insulin in in wire* medium (Ahmad A. et. al. (2015) ¹⁷ ). The low rate of insulin binding by the salt particles could be due to formation of a limited number of particles, whereas fast particle dissolution can also lead to unexpected premature insulin release from the degrading salt particles.

There remains a need for the development of an oral delivery agent for proteins and peptides that can overcome the multiple physico-chemical barriers of the GI tract while retaining sufficient bioavailability to generate an efficacious therapeutic response.

This invention thus aims to alleviate some or all of the problems of the prior art.

SUMMARY OF THE INVENTION

In accordance with an aspect of the invention, there is provided an oral delivery salt nano-precipitate for oral delivery of a protein molecule. The nano-precipitate comprises an inorganic metal salt capable of conferring a cation-rich domain to the nano-precipitate for electrostatic binding with the negative charges of a target protein molecule and an anion-providing salt capable of conferring an anion-rich domain to the nano-precipitate for electrostatic binding with the positive charges of a target protein molecule. Both the cation-providing and anion-providing salts are present in the nano-precipitate at a sufficiently high concentration to enable adequate saturation of cationic and anionic salts, per volume of the nano-precipitate, such that the target protein molecule is strongly bound therewith and buffered against premature release at an acidic pH.

The cation-providing inorganic metal salt may be a Group 2 metal salt.

The ratio of the cation-providing inorganic metal salt to the anion-providing salt may be 5:2.

The cation-providing inorganic metal salt may be a barium salt (Ba ²⁺ ), a strontium salt (Sr ²⁺ ), a calcium salt (Ca ²⁺ ), a ferrous salt (Fe ²⁺ ) or a zinc salt (Zn ²⁺ ).

The anion-providing salt may be a sulphate (SO4 ² )·

The anion-providing salt may be a sulphite (SO3 ² )·

The anion-providing salt may be a carbonate (CO3 ² )·

The nano-precipitate may be barium sulphate (BaSC> ₄ ).

The nano-precipitate may be barium sulphite (BaSCb).

The nano-precipitate may be barium carbonate (BaCCb).

In a second aspect of the invention, there is provided a method of producing the salt nano-precipitate of the invention. The method comprises the following steps:

(i) providing a volume of the cation-providing inorganic metal salt;

(ii) providing a volume of the anion-providing salt; and

(iii) mixing the salts of steps (i) and (ii) to form a salt precipitate.

The cation-providing inorganic metal salt may be a Group 2 metal salt. The Group 2 metal salt may be barium chloride (BaCL·).

The anion-providing salt may be sodium sulphate (Na ₂ SC> ₄ ). The anion-providing salt may be sodium sulphite (Na ₂ SC> ₃ ).

The anion-providing salt may be sodium carbonate (Na ₂ CC> ₃ ).

Protein molecule loading may be performed by adding a volume of protein molecules to the cationic metal salt solution of step (i), before addition of the anionic salt solution of step (ii), and, mixing the constituents until a precipitate is formed.

The protein-loaded salt nano-precipitates may be surface-modified with a bioadhesive protein. The bio-adhesive protein may be transferrin, casein or a folate binding protein.

The protein molecule loaded into the salt nano-precipitate may be insulin. The insulin-loaded salt nano-precipitate may be used in the treatment of hyperglycemia, type 1 or type 2 diabetes mellitus in an animal or a human subject. Such use may include oral administration of bicarbonate before treatment with the insulin-loaded salt nano-precipitates.

The present invention seeks to overcome the problems of the prior art by providing salt nano-precipitates having both cation (positive) and anion (negative) rich- domains conferred by an inorganic metal salt (cation -providing salt) and an anion providing salt. Due to the heterogeneous charge distribution, these salt nanoprecipitates are capable of binding protein molecules through electrostatic interactions.

In particular, the salt nano-precipitates of this invention are very effective carriers of protein molecules that carry both positive and negative charges such as insulin, peptide hormones (e.g. GLP-1), enzymes, cytokines and monoclonal antibodies. These protein molecules have positive and negative charges because of the amine and carboxylic groups that interact, respectively, with the anion and cation rich domains of the salt nano-precipitates. In fact, every protein molecule has a net charge of either negative or positive at physiological pH, which enables the protein to easily bind to cation or anion rich domains of the salt nano-precipitates of this invention. As explained in the previous section, the main technical barriers for oral delivery of protein molecules include rapid degradation of the protein molecule before arrival at the target site (due to pH ranging from basic to very acidic in the stomach and small intestine) as well as limited permeability of the intestinal lining for protein molecules (primarily due to the mucin barrier).

The salt nano-precipitates of this invention are able to overcome these barriers while retaining the efficacy of the protein molecule i.e. providing efficacious therapeutic bioavailability of the protein molecule in a subject. The cation and anion rich- domains of the salt nano-precipitates of this invention enable not only good protein loading but also resistance to premature release of the protein molecule before the target site. Presence of the cation-providing and anion-providing salts in the nanoprecipitate at a sufficiently high concentration to enable adequate saturation of cationic and anionic salts, per volume of the nano-precipitate is key to the strong binding of the target protein molecule and effective buffering against premature release at an acidic pH. This enables the protein molecule to be protected against degradation by harsh pH and enzymatic environments.

The heterogenous charge nature of the salt nano-precipitates of this invention also enables it to efficiently bind to mucin i.e. enable the protein molecule bound to the salt nano-precipitate of this invention to effectively overcome the mucin barrier, be absorbed through the intestinal wall and enter systemic blood circulation so as to be bioavailable.

There is no harsh temperature or pH required during the precipitation of and/or protein loading into the salt nano-precipitates of this invention. This is a distinct advantage as protein molecules are prone to degradation or inactivation in harsh temperatures or pHs. The synthesis conditions of the salt nano-precipitates of this invention can be easily modifiable depending on the protein molecule and target site of delivery. Modifying the synthesis conditions can also influence protein-loading capacity and the protein-release profile of the salt nano-precipitate.

Various other advantages of the oral delivery salt nano-precipitate of this invention will be further elaborated in the following pages. BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated, although not limited, by the following description of embodiments made with reference to the accompanying drawings in which:

Figure 1 is a simplified schematic representation of the precipitation reaction.

Figure 2 is a simplified illustration of insulin loading into the salt nano-precipitates.

Figures 3A, 3B and 3C show FT-IR spectrums of barium sulphate (BaSCU), barium sulphite (BaSC ) and barium carbonate (BaCOa), respectively.

Figures 4A, 4B and 4C show Energy Dispersive X-Ray Spectroscopy (EDX) analysis of barium sulphate (BaSC> ₄ ), barium sulphite (BaSC ) and barium carbonate (BaCOa), respectively.

Figure 5 show dissolution profiles of barium sulphate (BaS04), barium sulphite (BaSOa) and barium carbonate (BaCOa), upon exposure to pH 7.78 to pH 1.78 over a period of 3 hours.

Figure 6A shows the percentage of loading of FITC-insulin into BaS0 ₄ , BaSOa and BaCOa.

Figure 6B shows the percentage of release of FITC-insulin at lower phis (pH 7.14 to pH 1.78).

Figures 7A, 7B and 7C are FE-SEM micrographs of BaS0 ₄ , BaSOa and BaCOa particles, respectively.

Figures 8A, 8B, 8C, 8D and 8E are FE-SEM images of free BaS0 ₄ particles, low concentration (IX) BaS0 ₄ particles loaded with 2 pg, 10 pg, 50 pg insulin, and, high concentration (20X) BaS0 ₄ particles loaded with insulin, respectively. Figures 9A, 9B and 9C are FE-SEM images of free BaSCb particles, low concentration (IX) insulin-loaded BaSCb particles and high concentration (20X) insulin-loaded BaSC particles, prepared with 50 pg of insulin, respectively.

Figures 10A, 10B and IOC are FE-SEM images of free BaCCb particles, low concentration (IX) insulin-loaded BaCCb particles and high concentration (20X) insulin-loaded BaCCb particles, respectively.

Figures 11A, 11B and 11C are FT-IR spectrums showing mucin adhesion to BaSC> ₄ , BaSCb and BaCCb particles, respectively.

Figure 12 shows mucin adhesion efficiency of BaSC> ₄ , BaSCb and BaCCb particles.

Figure 13 is a graph showing the effects of oral delivery of free insulin versus insulin- loaded BaSC> ₄ , BaSC and BaCCb particles to STZ-induced diabetic rats.

Figure 14 are graphs showing the effect of oral administration of insulin-loaded BaSC> ₄ , BaSC and BaCCb particles to STZ-induced diabetic rats.

Figure 15 is a graph showing the effect on blood glucose levels of pre-administering bicarbonate before oral gavage of insulin-loaded BaSC> ₄ , BaSCb or BaCCb particles.

Figure 16 is a graph showing the effect on blood glucose levels of oral administration (a) only with insulin, (b) only with insulin-loaded BaSC> ₄ particles, and (c) with bicarbonate followed by insulin-loaded BaSC> ₄ particles.

Figure 17 show pictures of SDS-PAGE gels with different band intensities for albumin-bound BaSC> ₄ , BaSCb and BaCCb particles following Coomassie staining.

Figure 18 show the albumin band pictures of albumin-loaded BaSC> ₄ , BaSCb and BaCCh particles following exposure to trypLE enzyme in medium with pH 7.4, 6.8 and 1.8. Figure 19 is a graph showing the relative percentage of albumin degradation calculated from the band intensities.

Figure 20A show gel pictures of free albumin, followed by free albumin exposed to sGF and albumin-loaded BaSC> ₄ particles exposed to sGF.

Figure 20B show gel pictures of free albumin, followed by free albumin exposed to sGF and albumin-loaded BaSC particles exposed to sGF.

Figure 20C show gel pictures of free albumin, followed by free albumin exposed to sGF and albumin-loaded BaCC particles exposed to sGF.

Figure 21 is a graph showing the effect on blood glucose levels over a 6-hour period following oral administration of SrSC>3-insulin conjugation (200 IU/kg of insulin) after a 14-hour starvation.

Figure 22 is a graph showing the effect on blood glucose levels over a 6-hour period following oral administration of SrSC>3-insulin conjugation (100 IU/kg of insulin) after a 14-hour starvation.

Figure 23 is a graph showing the effect on blood glucose levels over a 6-hour period following oral administration of SrSC -insulin conjugation (50 IU/kg of insulin) after a 14-hour starvation.

Figure 24 is a graph showing the effect on blood glucose levels over a 6-hour period following oral administration of SrSC>3-insulin conjugation (100 IU/kg of insulin) without starvation.

Figure 25 is a graph showing the effect on blood glucose levels over a 6-hour period following oral administration of SrSC>3-insulin conjugation (100 IU/kg of insulin) after a 14-hour starvation.

Figure 26 is a graph showing the effect on blood glucose levels over a 6-hour period following intravenous administration of free insulin. Figure 27 is a graph showing the effect on blood glucose levels over a 6-hour period following oral gavage of insulin-loaded transferrin modified BaSC> ₄ .

Figure 28 is a graph showing the effect on blood glucose levels over a 4-hour period following oral gavage of insulin-loaded casein modified BaSC> ₄ .

Figure 29 is a graph showing the percentage of reduction in blood glucose levels over a 4-hour period following oral gavage of insulin-loaded casein modified BaSC> ₄ .

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention relates to an oral delivery salt nano-precipitate for oral delivery of protein molecules. The salt nano-precipitate of this invention mainly comprises a cation-providing inorganic metal salt and an anion-providing salt. The target protein molecule is bound to the nano-precipitate by way of electrostatic interactions.

The cation-providing inorganic metal salt

The cation-providing inorganic metal salt of this invention can be any suitable inorganic metal salt that is capable of conferring a cation-rich domain to the salt nano-precipitate for electrostatic binding with the negative charges of a target protein molecule.

The cation -providing inorganic metal salt may be any one of a barium salt (Ba ²⁺ ), a strontium salt (Sr ²⁺ ), a calcium salt (Ca ²⁺ ), a ferrous salt (Fe ²⁺ ) or a zinc salt (Zn ²⁺ ). Preferably, the cation-providing inorganic metal salt is a Group 2 metal salt.

In a preferred embodiment, the cation-providing inorganic metal salt is a barium salt (Ba ²⁺ ) that can be provided by way of any water-soluble barium salt such as barium chloride (BaCh).

The cation-providing inorganic metal salt must be present in the salt nanoprecipitate of this invention at a sufficient concentration to confer a cation-rich domain to the salt nano-precipitate for electrostatic binding with the negative charges of a target protein molecule. The cation-providing inorganic metal salt should be present in the salt nano-precipitate of this invention at a concentration of between about 1 mM to about 1 M, preferably, between about 10 mM to about 1 M, and, most preferably, between about 100 mM to about 1 M.

The anion-providing salt

The anion-providing salt of this invention can be any suitable salt that is capable of conferring an anion-rich domain to the salt nano-precipitate for electrostatic binding with the positive charges of a target protein molecule.

The anion-providing salt may be any one of a sulphate (SO4 ^2" ), a sulphite (SO3 ^2" ) or a carbonate (CO3 ^2" ) that can be provided by way of any water-soluble salt such as sodium sulphate (Na ₂ SC> ₄ ), sodium sulphite (Na2SC>3), and sodium carbonate (Na ₂ C0 ₃ ).

The anion-providing salt must be present in the salt nano-precipitate of this invention at a sufficient concentration to confer an anion-rich domain to the salt nano-precipitate for electrostatic binding with the positive charges of a target protein molecule. The anion-providing salt should be present in the salt nano-precipitate of this invention at a concentration of between about 1 mM to about 1 M, preferably, between about 10 mM to about 1 M, and, most preferably, between about 100 mM to about 1 M.

The salt nano- precipitate

The salt nano-precipitate of this invention is built on a concept of complexing a target protein molecule with inorganic salt particles on the basis of ionic interactions. Due to the heterogeneous charge distribution, the salt nano-precipitates of this invention are capable of binding target therapeutic protein molecules like insulin through electrostatic interactions. For example, insulin (5.8kDa) has localized surface charges (either positive or negative at basic or acidic pH). These surface charges of insulin allow it to electrostatically bind with either the cationic or anionic domains of the salt nano-precipitates, depending on the pH of the host solution.

The ratio of the cation-providing inorganic metal salt to the anion-providing salt in the salt nano-precipitate of this invention is preferably about 5 : 2. As demonstrated in the Examples at the end of the description, the inventors have surprisingly found that this ratio of cation -providing salt to anion-providing salt results in a high rate of particle formation. One possible explanation for this is that the cation-providing salts act as a powerful driving force to accelerate the precipitation reaction needed for particle formation.

For oral delivery of insulin, the inventors have surprisingly found that the preferred salt nano-precipitates of this invention are barium sulphate (BaS04), barium sulphite (BaSOa) and barium carbonate (BaCOa), with the most preferred compounds being barium sulphate (BaS04) and barium carbonate (BaCOa).

From field emission scanning electron microscopy (FE-SEM) analysis of the preferred salt nano-precipitates of this invention i.e. BaS0 ₄ , BaS0 ₃ and BaCOa, the inventors observed the following regarding the morphology of the precipitates in terms of particle size, shape and aggregation pattern.

Surprisingly, the inventors noticed that there was a distinct morphological difference between the low salt concentration (IX) and high salt concentration (20X) salt nanoprecipitates. This was true for all three Ba salt nano-precipitates of this invention i.e. BaSC> ₄ , BaSCh and BaCCh. Further details are provided in the Examples. Notably, it is postulated that the distinct morphology of the high concentration (20X) salt nano-precipitates is a pertinent factor in the improved efficacy of the high concentration salt nano-precipitates when compared to the low concentration salt nano-precipitates, in reducing blood glucose levels in test subjects (diabetic rats).

The low concentration (IX) BaSC> ₄ particles have a distinctive irregular, oval to hollow round shape. The larger particles have an oval to hollow round shape with a particle size of between 110 to 200nm and the smaller particles are irregular shaped with a particle size of between 30 to 100 nm. On the other hand, for the high concentration (20X) BaSC> ₄ particle formulation, only smaller round shape particles were observed.

The low concentration (IX) BaSCh particles ranged in size from large (about 90 to 270 nm) to very small (about 15 to 40 nm) sized particles and were found to be of a round shape and held in tight baseball-shaped clusters with a rough surface area. Each distinctive particle cluster ranged from about 500 nm to 1.0 pm in diameter. Larger particles displayed a rounder shape with smooth surface area without forming clusters. The particles in the high concentration (20X) BaSCh formulation were relatively less aggregated with formation of clusters that look irregular, with no change in single particle size and shape.

The low concentration (IX) BaCCh particles were observed to have a distinctive square to rectangular shaped morphology, with the particle size varying widely from about 50 to 500 nm. The BaCCh particles in the low concentration formulation were also observed to be closely aggregated with one another and had a tendency to form rod or filament-like structures. In the high concentration (20X) formulation, the BaCCh particle clusters formed long, thin threads that appeared to be inter- tangled with each other, with no change in single particle morphology.

One of the biggest challenges in oral delivery of protein therapeutics is pre-systemic degradation due to fluctuating pH conditions, for example, extremely acidic (~ pH 1) pH in stomach when patient is in a fasting state. Hence, any oral delivery agent needs to be resistant to extreme acidic pH to survive transport through the gastrointestinal system.

From in vitro testing of the preferred salt nano-precipitates of BaS04, BaS03 and BaC03, the inventors observed that the BaS04 particles demonstrated an excellent resistance to degradation over a wide range of pH (from pH 1 to pH 7.78) over a 3- hour period. BaCCh also maintained almost the same level of resistance to degradation throughout the range of pH, with no significant particle loss at low pH. However, the inventors observed that BaSCb particles experienced significant degradation at pH 1.69. Resistance to both basic and acidic pHs through a period of 3 hours (ideal stomach and intestinal residence time) demonstrates that the salt nano-precipitate of this invention can withstand the harsh gastrointestinal pH and enzymatic environment and protect the target protein molecule against pH and enzymatic degradation.

In addition to resistance to pH-induced degradation, an ability to efficiently bind with mucin is also an important indicator of an oral delivery agent's capacity for intestinal absorption. Mucins are highly O-glycosylated molecules that have gel-like properties and play an important role in protecting the intestines from luminal digestive enzymes, abrasion by food particles, and pathogens (i.e. chemical or physical injury) by forming a barrier between the lumen and the intestinal epithelium. In order to be absorbed into the intestinal lumen, oral delivery agents containing target protein therapeutics need to successfully pass through the mucin barrier.

Following in vitro tests (Fourier Transform Infrared Spectroscopy (FT-IR) and spectrophotometric protein quantification), the inventors observed that the preferred salt nano-precipitates of BaSC> ₄ , BaSCh and BaCCh did indeed have an affinity to mucin and would therefore be capable of crossing the intestinal lining.

FT-IR analysis confirmed mucin binding by all of BaSC> ₄ , BaSC>3, BaCCh.

Mucin-adhered BaSC> ₄ particles demonstrated 12 distinctive peaks (see Figure 11A), with peaks 2360 cm ^-1 , 1188 cm ^-1 , 1064 cm ^-1 , 982 cm ^-1 , 635 cm ^-1 and 605 cm ^-1 matching the peaks of mucin-free BaSC> ₄ particles in similar positions. Peak 3415 cm ^-1 showed a deviation to 2392 cm ^-1 probably due to mucin attachment. Similarly, mucin peaks at 3279 cm ^-1 , 1549 cm ^-1 and 1434 cm ^-1 were observed to be shifted to 3275 cm ^-1 , 1539 cm ^-1 and 1439 cm ^-1 , respectively, in mucin-adhered BaSC> ₄ particles. Existence of characteristic peaks for BaSC> ₄ and mucin indicates adherence or binding of BaSC> ₄ particles to mucin.

Mucin-adhered BaSC particles showed 13 distinctive peaks (see Figure 11B). The peaks of BaSCh particles, 1133 cm ^-1 , 912 cm ^-1 , 630 cm ^-1 and 409 cm ^-1 were found at 1131 cm ^-1 , 919 cm ^-1 , 509 cm ^-1 and 474 cm ^-1 positions in mucin-adhered BaSCh particles. On the other hand, 8 characteristic peaks from mucin were found at 3311 cm ^-1 , 1648 cm ^-1 , 1539 cm ¹ , 1434 cm ¹ , 1298 cm ¹ , 1343 cm ¹ , 1187 cm ¹ and 1101 cm ¹ positions. Mucin peaks showed deviation from their positions due to binding to BaS0 ₃ particles. The presence of characteristic peaks for BaS0 ₃ as well as for mucin indicates positive adhesion of BaS0 ₃ particles to mucin.

Mucin-adhered BaC0 ₃ particles demonstrated 9 distinctive peaks (see Figure 11C). Mucin characteristic peaks at 3279 cm ¹ , 1634 cm ¹ , 1549 cm ¹ , 1232 cm ¹ , 1115 cm ¹ were found respectively at 3384 cm ¹ , 1645 cm ¹ , 1555 cm ¹ , 1213 cm ¹ , 1121 cm ¹ positions in mucin-adhered BaCCb particles. The presence of characteristic peaks for Ba(X> ₃ particles and mucin indicates positive adhesion of BaCC> ₃ particles to mucin.

The percentage of mucin adhesion to BaSC> ₄ , BaSC , BaCCb was calculated by measuring the protein content (mucin) precipitated out with the particles after centrifugation. Following protein quantification by the Bradford method, the inventors observed that BaSC> ₄ particles showed the highest mucin adhesion (100%), followed by BaSC and BaCCb particles with about 60 to 70% mucin adhesion. Such high mucin adhesion (i.e. the salt nano-precipitates of this invention has the ability to efficiently bind to mucin) facilitates the uptake of the salt nano-precipitates across the intestinal epithelium for intestinal absorption of the target protein molecule.

Synthesis of empty salt nano-precipitates

The salt nano-precipitates of this invention can be prepared by firstly, providing a volume of the cation-providing inorganic metal salt solution. A volume of the anionproviding salt solution is separately provided.

The two volumes of salt solution are then mixed at a pre-determined volume ratio of 5 parts of the cation-providing inorganic metal salt solution to 2 parts of the anionproviding salt solution. The solution is mixed until homogenous and incubated for 30 minutes at 37°C for formation of the salt precipitate.

There is no harsh temperature or pH required during the precipitation of and/or subsequent protein loading into the salt nano-precipitates of this invention. This is a distinct advantage as protein molecules are prone to degradation or inactivation in harsh temperatures or pHs.

The synthesis conditions of the salt nano-precipitates of this invention can be easily modifiable depending on the protein molecule and target site of delivery. Modifying the synthesis conditions can also influence protein-loading capacity and the protein- release profile of the salt nano-precipitate. For example, modifying the salt particles with an organic molecule, such as citrate or alpha ketoglutarate, which carry carboxylic groups that can bind to the cations of the particles, can influence the binding and release of proteins.

Loading of target protein molecule into salt nano- precipitates

Protein loading may be performed by adding a volume of the target protein molecule to the cationic-providing inorganic metal salt solution. Subsequently, the anionicproviding salt solution is added to the mixture.

The solution is mixed until homogenous and incubated for 30 minutes at 37°C for formation of the salt precipitate.

During in vitro testing, the inventors observed that the salt nano-precipitates have a high affinity with protein molecules.

For example, when assessing the affinity of insulin molecules towards barium sulphate (BaSC^), barium sulphite (BaSCh) and barium carbonate (BaCOa) in vitro, the inventors observed that BaCOa showed the highest binding affinity for insulin (100% binding affinity) when compared to BaS0 ₄ and BaSOa.

Further, the inventors observed that when the insulin-loaded BaS0 ₄ , BaSOa and BaCOa were exposed to a wide range of pH in vitro, nearly 20%, 50% and 60% of insulin were released from BaS0 ₄ , BaCOa and BaSOa, respectively, when the respective insulin-salt precipitate complexes were exposed to pH of 2.47. At a pH of 1.78, only around 30% of insulin was released from the BaS0 ₄ -insulin complexes and approximately 80% of insulin was released from the BaS0 ₃ -insulin complexes. This means that both BaSC> ₄ and BaSC can protect insulin, to an extent, from degradation in stomach acidic pH, but that BaSC> ₄ is more effective than BaSC in preventing insulin from degradation.

From FE-SEM analysis of insulin-loaded BaSC> ₄ , BaCC and BaSC , the inventors observed various changes in morphology of the insulin-loaded precipitates versus the empty precipitates i.e. insulin loading had clearly altered the shape of the nanoprecipitates with different concentration of insulin resulting in different degree of structural changes in morphology.

At a low concentration of insulin (e.g. 2 pg), insulin-loaded BaSC> ₄ particles were observed to be morphologically distinct from the empty particles in that the insulin- loaded particles are baseball shaped with a rough surface area and without a hollow structure. In contrast, as mentioned above, empty BaS04 particles have a round shape with a distinctive hollow structure in the middle and a smooth outer surface. At increasing concentrations of insulin (e.g. 10 pg and 50 pg), the insulin-loaded BaSC> ₄ particles looked more elongated and the surface area appeared rougher. Notably, there were no empty BaSC> ₄ particles observed at high concentrations of insulin.

For the BaSCh particles, no noticeable changes of size, shape or surface morphology were observed between the insulin-loaded and the empty particles. However, the inventors observed very distinctive changes in aggregation pattern. As mentioned above, the empty BaSCh particles tended to clump together whereas the insulin- loaded particles were much less prone to aggregation, with their clusters clearly oval shaped.

Much like the BaSCh particles, there were also no noticeable changes of size, shape and surface morphology of insulin-loaded and empty BaCCh particles. Once again, the inventors observed very distinctive changes in aggregation patterns. While the empty BaC03 particles tended to clump together, the insulin-loaded BaC03 particles were much less prone to aggregation. In an embodiment, the protein-loaded salt nano-precipitates can also be surface- modified with bio-adhesive proteins such as transferrin, casein and/or folate binding proteins. The inventors have demonstrated in the following Examples that doing so can help in increasing intestinal absorption of the protein-loaded precipitates, which in turn leads to increased absorption into the blood stream i.e. improved bioavailability.

Therapeutic use of insulin-loaded salt nano-precipitates

The salt nano-precipitates of this invention are suitable for use in the oral delivery of protein therapeutics to animal or human subjects.

For example, insulin-loaded salt nano-precipitates such as barium sulphate (BaSCX , barium sulphite (BaSCb), barium carbonate (BaCCb) and strontium sulphite (SrSC ) are suitable for treating hyperglycemia, type 1 or type 2 diabetes mellitus in an animal or a human subject.

During laboratory testing on an animal subject (streptozotocin (STZ)-induced diabetic male Wister Kyoto (WKY) rats), the inventors observed that all treatment groups of diabetic rats orally fed with insulin-loaded particles of BaSC> ₄ , BaSCb, BaCCh and SrSCh, respectively, showed a reduction in blood glucose levels within 1 hour of oral administration. By comparison, diabetic WKY rats orally fed free insulin did not show any significant reduction of blood glucose levels at any point in time during a 4-hour long observation.

The inventors noticed during initial in vivo testing that a high salt concentration (20X) formulation of the salt nano-precipitates of this invention is distinctly more efficacious in reducing blood glucose levels in test subjects (diabetic rats) in comparison with a low salt concentration (IX) formulation, when orally administered. This could be because a 20X concentration formulation contained more adequate saturation of salt particles per volume, to more strongly bind insulin and to more efficiently act as a buffering agent in the acidic stomach environment. As mentioned in a preceding section of the description, there is a distinct morphological difference between the low salt concentration (IX) and high salt concentration (20X) salt nano-precipitates. It is possible that the distinct morphology of the high concentration (20X) salt nano-precipitates also plays a part in the improved efficacy of the high concentration salt nano-precipitates in reducing blood glucose levels.

All in vivo tests explained below and in the Examples were conducted with the high salt concentration (20X) formulation.

For the group orally fed with insulin-loaded BaSC> ₄ particles, it was observed that the blood glucose level was significantly lowered compared to baseline levels within a time period of 1 to 3 hours (p<0.05). At the 4 ^th hour, the blood glucose level was still low relative to baseline level, but not significantly so and an ascending trend began to be observed.

For the group orally fed with insulin-loaded BaCCb particles, it was observed that there was a significant decrease of blood glucose levels within 1 to 2 hours. Flowever, blood glucose levels started to show a slight increase at the 3 ^rd hour, and by the 4 ^th hour, it was back to baseline levels.

For the group orally fed with insulin-loaded BaSC particles, a reduction in blood glucose levels was observed but the reduction was not significant at the 4-hour mark.

For the group orally fed with insulin-loaded SrSCh particles, it was observed that there was a significant decrease in blood glucose levels in the beginning (even with a relatively low concentration of insulin 50 IU/kg), and this continued to be observed beyond the 4-hour mark.

Significantly, the inventors observed that the effect on blood glucose levels generated during oral administration of the above-mentioned insulin-loaded salt nano-precipitates of this invention was surprisingly similar to the effect generated by subcutaneous delivery of commercial human insulin aspart (Novorapid, Novonordisk). Insulin aspart, when administered subcutaneously, generally only lasts 4 to 5 hours, with a maximum drop in blood glucose levels during the 2 to 3- hour period.

As demonstrated in the following Examples, the inventors also surprisingly observed that the oral administration protocol that yields the best results with regards to reducing blood glucose levels involves pre-administration of bicarbonate orally before oral treatment with the insulin-loaded salt nano-precipitates of this invention. This is likely because the bicarbonate generated an elevated stomach pH level in the test subjects (diabetic rats), which resulted in an improved pH environment for passage of the salt nano-precipitates and subsequent absorption through the intestinal epithelium.

Oral delivery forms of protein-loaded salt nano-precipitates

The protein-loaded salt nano-precipitates of this invention may be processed into a suitable oral delivery dosage form, preferably, an oral liquid dosage form such as an oral suspension or an oral mixture, and, most preferably, an oral suspension.

The oral liquid dosage form may have a concentration of the protein-loaded salt nano-precipitates of this invention suspended in a suitable inert conventional carrier and/or diluent.

The protein-loaded salt nano-precipitates should be present in the oral liquid dosage form in a therapeutically suitable concentration of at least about 1 mg/ml and preferably, about 1 gm/ml.

A therapeutically suitable concentration is taken to mean a concentration of the protein-loaded nano-precipitates sufficient to achieve therapeutic effect when orally administered to an animal or a human subject

For example, when preparing an insulin-loaded salt nano-precipitate for oral delivery, a therapeutically suitable concentration means a concentration sufficient to achieve therapeutic effect when orally administered to an animal or a human subject suffering from hyperglycemia, type 1 or type 2 diabetes mellitus.

Hyperglycemia is defined as an excessively high blood glucose level, either in a fasting state (100 to 125 mg/dl) or in a non-fasting state (> 180 mg/dl). Type 1 diabetes mellitus is defined as a form of diabetes in which very little or no insulin is produced by the pancreas resulting in high blood glucose levels. Type 2 diabetes mellitus is defined as a fasting blood glucose level exceeding 125 mg of glucose per dl of plasma.

As shown in the following Examples, the inventors have observed that a wide range of insulin concentrations can be loaded into the salt nano-precipitates of this invention, for example, from low concentration to high concentration e.g. from 1 to 100 IU/kg (IU/kg means 1 unit of insulin per kg of animal body weight).

EXAMPLES

The following Examples illustrate the various aspects of a salt nano-precipitate of this invention. These Examples do not limit the invention, the scope of which is set out in the appended claims.

Example 1: Synthesis of empty BaS04, BaS03 and BaC03 nano- precipitates

This example illustrates the synthesis of empty BaSC> ₄ or BaSCh or BaCCh nanoprecipitates.

Dulbecco's Modified Eagle's Medium (DMEM) powder was purchased from Invitrogen. Hydrochloric acid (HCI) (1M), sodium hydrogen carbonate (NaHCOa), barium chloride dehydrate (BaCh.2H ₂ 0), sodium sulphite (Na ₂ SC> ₃ ) and mucin were bought from Sigma Aldrich. Additionally, fluorescein isothiocyanate (FITC)-labelled insulin stock (human, recombinant, expressed in yeast, lyophilized powder) was purchased from Sigma Aldrich. HEPES (2-(4-(2-hydroxyethyl)-l- piperazinyl)ethanesulfonic acid) and sodium carbonate (Na2CC>3) were from Fisher Scientific and sodium sulphate (Na ₂ SC> ₄ ) from Merck. Insulin aspart (NovoRapid®, Novo Nordisk) was purchased from a local pharmacy. Pepsin was purchased from Promega (USA).

1M of BaCh, Na2SC>4, Na2SC>3 and Na2CC>3 stock solutions were prepared by calculating amount of the respective molecular weight of powder and dissolving them in water. All solutions were stored in 1 mL aliquots at -20°C. lOOmL of bicarbonated DMEM solution was freshly prepared by dissolving 1.35g of DMEM powder in 95 mL pure Milli-Q water, followed by the addition of 0.37g of sodium hydrogen carbonate (0.44 mM final concentration). The pH of the solution was then adjusted to the desired level by addition of either 1M HCI or 1M NaOH. Final volume was then adjusted to lOOmL. 2mg/mL of FTTC-insulin stock solution was prepared by dissolving the FITC-insulin powder into an appropriate volume of pure Milli-Q® water. Each Ba salt precipitate (BaSC> ₄ , BaSCL or BaC0 ₃ ) was prepared by incorporating a volume of cation-providing BaCh salt into either a volume of HEPES-buffered solution (pH adjusted to 8.0) or milliQH20 and mixing the resultant solution with a volume of one of an anion-providing salt i.e. Na ₂ SC> ₄ , Na2SC>3 or Na2CC>3, respectively. The final mixture was incubated for 30 minutes at 37°C and subsequently added to miliQH20 to obtain the final volume of particle suspension.

Table 1 below shows the various concentrations of reacting salts used to synthesize the three different Ba salt precipitates. Table 1: Various concentrations of reacting salts for synthesis of BaS04, BaS03 or BaC03

Figure 1 shows a simple diagrammatic representation of the precipitation reaction. Example 2: Insulin loading into BaS0 ₄ , BaS0 ₃ and BaC0 ₃ nano-precipitates

This example illustrates protein (insulin) loading of BaSC> ₄ or BaSCb and BaCCb nanoprecipitates.

Three Ba salt precipitates (BaSC> ₄ , BaSCh or BaCCh) were prepared as per Example 1 i.e. by incorporating a volume of cation-providing BaCh salt into either a volume of HEPES-buffered solution (pH adjusted to 8.0) or miliQH20 and mixing the solution with a volume of one of an anion-providing salt i.e. Na ₂ SC> ₄ , Na2SC>3 and Na2CC>3, respectively. Insulin was introduced right before addition of the second salt to the medium. The final mixture was incubated for 30 minutes at 37°C and subsequently added to milliQH20 to obtain the final volume of particle suspension.

Table 2 below shows the various concentrations of reacting salts and insulin used for preparation of the insulin-loaded Ba salt precipitates.

Table 2: Various concentrations of reacting salts and insulin to prepare insulin-loaded BaSOt, BaS(¾ or BaC03 The 20X concentration (high salt concentration) Ba salt precipitates prepared as per Table 2 above were used in all the following in vivo testing Examples.

Figure 2 shows a simplified diagrammatic depiction of insulin loading of the Ba salt precipitates. Example 3: Characterization and morphological screening of BaS0 ₄ , BaS0 ₃ and BaC03 nano-precipitates

Fourier Transform Infrared Spectroscopy (FT-IR)

A volume of 250 mI of 1M of BaCh was taken in a centrifuge tube, followed by addition of 100 mI of 1M Na ₂ SC> ₄ or Na ₂ SC> ₃ or Na ₂ CC> ₃ to the respective tubes to prepare BaSC> ₄ , BaSC and BaCC precipitates. The samples were then incubated at 37°C for 30 minutes. The final volume of the solution was made to 50 ml with addition of milliQH20. Samples were then centrifuged for 15 minutes at RPM 5,000. Precipitates were separated from the supernatant, stored sequentially at -20°C overnight and at -80°C for 0.5 hour and finally placed into a freeze-dryer (Labconco freeze dryer, Kansas City, MO, USA) for 5 hours. Samples were read using a Varian FT-IR (Santa Clara, CA, USA) and analysed with Resolution Pro 640 software.

FT-IR was used to confirm formation of the Ba salt precipitates BaS0 ₄ , BaS0 ₃ and BaCOs) by revealing the functional groups. Figure 3 shows the FT-IR spectrum obtained for the three different Ba salt precipitates.

The IR spectrum for BaS0 ₄ (Fig. 3A) shows peaks at 1188cm ¹ , 1060cm ¹ and 982cm ¹ , which are attributed to symmetric stretching of SO4 ^2" , peaks at 1635cm ¹ due to stretching vibration of SO4 ^2" and peaks at 604cm ¹ and 637cm ¹ , representing out-of-plane bending for SO4 ^2" . Peaks at 2360cm ¹ seem to be the overtone of S-0 vibration (Sifontes A. B. et. al. (2015) ¹⁷ , Ramaswamy V. et. al. (2010) ¹⁸ ). Existence of the functional group suggests the generation of BaSC> ₄ particles.

Fig. 3B shows the characteristic peaks for SO3 ^2" found at 490cm ¹ , 630cm ¹ , 912cm ¹ and 1133cm ¹ position, suggesting the formation of BaSCh particles (Qiao X. et. al. (2018) ¹⁹ ).

The characteristic peaks for CO3 ² found at 855cm ¹ and 692cm ¹ (Fig. 3C) were owing to in-plane and out-of-plane bending, whereas the peak at 1414cm ¹ corresponds to the asymmetric stretching of C-0 bond and peak at 1059cm ¹ is attributed to the symmetric C-0 stretching vibration (Sreedhar B. et. al. (2012) ²⁰ ), thus indicating the formation of BaCCb particles.

Elemental analysis of particles by Energy Dispersive X-Ray Spectroscopy (EDX)

5mI of cation- providing salt was added to 20 pi milliQH20, followed by addition of 2 mI of anion-providing salt. After 30 minutes of incubation at 37°C, 1 ml of milliQ H2O was added. 2 mI of the solution containing the particles was transferred on a coverslip. The coverslip was then air-dried in elevated temperature (45°C) inside a dryer for 1 hour. The dried sample was marked with a circular line with a marker for easy identification under a microscope. Samples were then subjected to platinum sputtering for 45 seconds with 30mA and factor of 2.3. Visualizations and documentation were done using a Field Emission Scanning Electron Microscope (FE- SEM) (Hitachi/SU8010, and Tokyo, Japan) at 5.0 kv.

EDX was carried out for elemental analysis of Ba salt particles (Figure 4). The presence of the desired elements in the samples confirm formation of the respective particles. Elemental analysis of BaSC> ₄ (Figure 4A) and BaSCb (Figure 4B) shows the presence of Ba, S and O, whereas analysis of BaCCb confirms the presence of Ba, C and O (Figure 4C).

Example 4: Particle stability assessment of BaS0 , BaS0 ₃ and BaC03 nanoprecipitates

One of the biggest challenges in oral delivery is pre-systemic degradation due to harsh pH in the stomach environment, which has a fluctuating pH and can be extremely acidic (~ pH 1) when the subject is in a fasting state. Any oral delivery agent needs to be resistant to extreme acidic pH to survive gastrointestinal transport. A set of in vitro experiments were designed and carried out to test the stability of the BaS04, BaS03 and Ba(X>3 particles in different phis, by exposing the formulations to a wide range of phis for a period of 3 hours. BaSC> ₄ , BaSCb and BaCCb nano-precipitates were prepared as per Example 1. After 30 minutes of incubation at 37°C, 1 ml of DMEM of different pHs (pH 7.78 to pH 1.78) was added to each sample and the samples were then kept at 37°C. The samples were prepared in triplicates and measured at 320 nm at different time points over a period of 3 hours.

Figure 5 shows changes in the turbidity pattern at 320nm for all three Ba salt particles. Both BaSC> ₄ and BaSCb particles demonstrated much higher absorbance than BaCCb particles when measured immediately after their generation at pH 7.78, reflecting the tendency of BaSCb and BaSCb to generate significantly higher number of particles than BaCCb.

Consistently high turbidity values for BaSCb particles indicate their excellent resistance to degradation over a wide range of pHs at different time points (1 to 3 hours), while the turbidity of BaSCb particles sharply dropped at pH 1.69 suggesting a considerable amount of particle loss. BaCCb maintained almost the same level of turbidity throughout the range of pHs, with apparently no significant particle loss at low pH.

Example 5: Insulin loading efficiency of BaS0 ₄ , BaS0 ₃ and BaC0 ₃ nanoprecipitates

This example illustrates the protein (insulin) loading efficiency of BaSCb or BaSCb and BaCCb nano-precipitates.

FrTC-insulin-loaded BaSCb, BaSCb and BaCCb were prepared as per Example 2. After 30 minutes of incubation at 37°C, 200 pi of DMEM prepared at different pHs (pH 7.78 to pH 1.78) was added. After incubation at room temperature for 10 minutes, the samples were centrifuged at 6000rpm for 2 minutes at 4°C. The supernatant was discarded and the resultant pellet was washed with 50 pi of HEPES, 2X. At the end of washing, the supernatant was again discarded and the pellet carrying insulin- bound particles was re-suspended with 200 pi of lOmM EDTA. 200 pL of supernatant was carefully transferred into a black 96-well plate (PerkinElmer Opti- Plate™-96 F) and fluorescence intensity values were recorded using a fluorescence micro-plate reader (PerkinElmer Victor X5 2030 Multi-label Reader) set with excitation/emission filters at 485nm/535nm wavelengths. A standard curve was prepared from absorbance values for known amounts of free FTTC-Insulin (0, 400, 800, 1200, 1600, 2000, 4000 qg) added to DMEM. % binding at pH 7.78 was calculated from the formula given below.

Percentage of release at subsequent pHs (7.18 to 1.78) was calculated by subtracting the amount of FTTC-insulin found in the pellet from the amount of FITC- insulin added initially and finally, multiplying by 100.

The affinity of insulin molecules towards BaSC> ₄ , BaSCh and BaCCh was assessed by separating FTTC-conjugated insulin-loaded particles from free fluorescent insulin by centrifugation. Fluorescence intensity was measured at 485nm/535nm (excitation/emission) wavelengths. The amount of FITC- insulin present in the pellet was calculated from the standard curve prepared with known amount of FTTC-insulin versus respective absorbance. The percentage of loading was calculated by dividing the amount of FITC-insulin present in pellet with the amount initially added and then multiplying with 100. Figure 6A shows the percentage of insulin binding to BaSC> ₄ , BaSCb and BaCC> ₃ . BaCCh particles demonstrated a higher binding affinity for insulin (100%) than BaSC> ₄ and BaSC particles. The equations used to calculate the percentage of loading and percentage of release of insulin are shown below. FrrC- insulin-loaded particles were exposed to a wide range of pHs, and as shown in Figure 6B, nearly 20%, 50% and 60% of insulin were released from BaSC> ₄ , BaCCb and BaSC particles, respectively, when the respective insulin-particle complexes were exposed to pH of 2.47. At a pH of 1.78, only around 30% of insulin was released from BaSC> ₄ and approximately 80% of insulin was released from BaSCb particles.

Morphological assessment of empty and insulin-loaded particles by FE- SEM

BaSCb, BaSCb and BaCCb were similarly prepared as described in Example 3 for FE- SEM and EDX analysis. Insulin-loaded salt particles were prepared separately (as per Example 2). Visualization and documentation were done using FE-SEM (Hitachi/SU8010, Tokyo, Japan) at 2.0 kv.

FE-SEM analysis was carried out to study the particle morphology with respect to particle size, shape and aggregation pattern with and without insulin loading into the salt particles.

Figure 7 shows micrographs for BaSCb, BaSCb and BaCCb. The BaSCb particles (Figure 7A) possessed distinctive irregular, oval to hollow rounder shape, with the bigger particles having oval to hollow rounder shape with particle size between 110 to 200nm and the smaller ones being irregular shaped with particle size between 30 to lOOnm. The BaSCb particles (Figure 7B) also had the prominent distribution of big (90 to 270nm) and very small (15 to 40nm) sized particles. The particles were found to be rounder shaped and held in tight baseball shaped clusters with rough surface area. Each distinctive particle cluster ranged from 500nm to l.Opm in diameter. Larger particles displayed a rounder shape with smooth surface area, without forming a cluster. The BaCCb particles (Figure 7C) demonstrated a distinctive square to rectangular shaped morphology, with the particle size varying widely from 50 to 500nm. The particles were closely aggregated with one another, forming rod or filament like structures. Figures 8 to 10 show the scanning electron microscopy images of empty BaSC> ₄ , BaSC and BaCC particles and insulin-loaded BaSC>4, BaSC and BaCC particles (IX low salt concentration and 20X high salt concentration, as per Table 2).

Effect of insulin loading into BaSCL particles

Insulin was loaded into the BaSC> ₄ particles in varying amounts. Loading of insulin clearly altered shape of the particles, with different concentrations of insulin showing different degrees of structural changes in particle morphology (Figure 8).

Figure 8A shows that free BaSCL particles are of a rounder shape with a distinctive hollow structure in the middle and an apparently smooth outer surface. Figure 8B shows BaSC> ₄ particles prepared with 2pg of initially added insulin, giving the impression of free particles residing with insulin-loaded particles which were morphologically different from the empty particles. Insulin-loaded particles are baseball shaped with a rough surface area and without a hollow structure. Figures 8C and 8D show insulin-loaded BaSCL particles prepared with 10pg and 50pg of insulin. With increasing insulin concentrations, the particles looked more elongated and the surface area appeared rougher. Interestingly enough, there was no empty BaSCL particle visualized at high concentrations of insulin.

Figure 8E shows high salt concentration (20X) insulin-loaded BaSC> ₄ particles. As can be seen, the high salt concentration particles are morphologically distinct from low salt concentration particles (Figures 8B, 8C and 8D) with only smaller round shape particles observed.

Effect of insulin loading into BaSC particles

Figure 9 shows the morphological features of empty and insulin (FITC-insulin)- loaded BaSCh particles. There were no noticeable changes in terms of size, shape or surface morphology. Flowever, very distinctive changes in aggregation pattern were observed. Empty particles tended to clump together whereas insulin-loaded particles were much less aggregated, with their clusters clearly oval shaped. From Figure 9C, it can be seen that high salt concentration (20X) insulin-loaded BaSCb particles are morphologically distinct from the low salt concentration particles (Figure 9B) with relatively less aggregation observed and formation of clusters that look irregular.

Effect of insulin loading into BaCC particles

Figure 10 shows morphological features of empty and insulin-loaded BaCC particles with no noticeable changes in size, shape and surface morphology of individual particles. Flowever, very distinctive changes in aggregation pattern were observed. Free particles tended to clump together whereas insulin-loaded particles looked much less aggregated.

Figure IOC shows that high salt concentration (20X) insulin-loaded BaCC particles are morphologically distinct from the low salt concentration particles (Figure 10B) with long, thin threads that appear to be inter-tangled with each other, observed.

Example 6: Assessment of adhesion of BaS04, BaS0 ₃ and BaC03 nanoprecipitates to mucin

This example illustrates the adhesion of BaSC> ₄ or BaSCh and BaCCh nanoprecipitates to mucin.

Spectrophotometric analysis of particle adhesion to mucin

The BaSC> ₄ , BaSCh and BaCCh particles were prepared as per Example 1. 200pl of milliQF O was added followed by addition of IOOmI of mucin (3g/L). The mixture was then incubated at 37°C for 10 minutes followed by centrifugation at RPM 13, for 5 minutes. The supernatant was collected in a fresh tube. 250mI of Bradford agent was directly added to the tube. The mixture was then incubated for 5 minutes before transferring to 96 well microplates.

Absorbance was read at 595nm using victor X5 spectrophotometer (PerkinElmer, USA). A standard curve was created using a series of different concentrations of mucin solution of 25 to 600pg, mixed with 250mI of Bradford agent. Unknown amount of mucin present in the supernatant was calculated from the standard curve. The percentage of adhesion of Ba salt particles to mucin was calculated from added mucin with the two-step formula shown below. mucm bound to particles x 100

%adhmkm “ added mucin

FT-IR assessment of BaS0 ₄ , BaS0 ₃ and BaC0 ₃ adhesion to mucin

In vitro tests were done to determine the affinity of BaSC> ₄ , BaSC and BaCC particles to mucin in order to predict whether the particles would be capable of crossing the intestinal lining. The data from FT-IR analysis would indicate any possible interactions of mucin molecules with the particles.

BaSC> ₄ , BaSC and BaCC samples were prepared in bigger volumes for this experiment. 250pl of 1M of BaCL· in a 50ml tube was mixed with IOOmI of 1M Na2SC>4 or Na2SC>3 or Na2CC>3 to prepare BaSC>4 or BaSCb or BaCC particles. The samples were then incubated at 37°C for 30 minutes. Sample tubes were then topped up to 25ml with addition of milliQH ₂ 0, followed by addition of a solution containing 5ml of mucin (3g/l). Samples were centrifuged for 15 minutes at 5,000 RPM. Precipitates were then stored at -20°C overnight, followed by storing at -80°C for 0.5 hour and freeze-drying (Labconco freeze dryer, Kansas City, MO, USA) for 5 hours. Varian FT-IR (Santa Clara, CA, USA) and Varian Resolution Pro 640 software (Santa Clara, CA, USA) were used to check the spectra of particles alone, particle- mucin complexes and mucin alone.

FT-IR was used to confirm mucin binding of BaS0 ₄ , BaS0 ₃ and BaC0 ₃ particles by revealing the functional groups for each particle along with the mucin protein. Samples for particles only, mucin only and particles adhered to mucin were analyzed with FT-IR. The comparison of the peak patterns showed positive mucin binding for BaSC> ₄ , BaSC and BaCCb particles. Free mucin displayed characteristic peaks of N-H at 3279cm ^-1 , Amide I at 1634cm ¹ , Amide II at 1549cm ¹ , C-H at 1434cm ¹ and 1374cm ¹ , Amide III 1232cm ¹ , C-O-C at 1115cm ¹ and C-C-0 at 1029cm ¹ (Liu F. et. al. (1988) ²² ).

Figure 11A shows the FT-IR spectrum of BaSC> ₄ particles alone, free mucin and BaSCL particle-mucin complexes. BaSC> ₄ particles showed 8 characteristic peaks, as described above in Example 3. Mucin displayed 8 characteristic peaks (as stated above). Mucin-adhered BaSC> ₄ particles demonstrated 12 distinctive peaks, with peaks 2360cm ¹ , 1188cm ¹ , 1064cm ¹ , 982cm ¹ , 635cm ¹ and 605cm ¹ matching the peaks of BaSC> ₄ particles in the similar positions. The peak at 3415cm ¹ showed a deviation to 2392cm ¹ probably due to mucin attachment to the particles. Similarly, mucin peaks at 3279cm ¹ , 1549cm ¹ and 1434cm ¹ seemed to be shifted to 3275cm ¹ , 1539cm ¹ and 1439cm ¹ respectively in mucin-attached particles. Existence of characteristic peaks for BaSC> ₄ and mucin indicates interactions between BaSC> ₄ particles to mucin.

Figure 11B shows the FT-IR spectrum for free BaSCb particles, free mucin and BaSC>3-mudn complex. BaSCb particles possess 4 characteristic peaks as described in Example 3. Mucin has 8 characteristic peaks (as mentioned above). Mucin- adhered BaSCb particles showed 13 distinctive peaks. The peaks of BaSCb particles, 1133cm ¹ , 912cm ¹ , 630cm ¹ and 409cm ¹ were found at 1131cm ¹ , 919cm ¹ , 509cm ¹ and 474cm ¹ positions in mucin-adhered BaSCb particles. 8 characteristic peaks from mucin were found at 3311cm ¹ , 1648cm ¹ , 1539cm ¹ , 1434cm ¹ , 1298cm ¹ , 1343cm ¹ , 1187cm ¹ and 1101cm ¹ positions. Mucin peaks showed deviation from their positions, likely due to its binding to BaSCb particles. The presence of characteristic peaks for BaSCb particle as well as for mucin indicates positive adhesion of BaSCb particles to mucin.

Figure 11C shows the FT-IR spectrum for free BaCCb particles, free mucin and BaCCb-mucin complex. BaCCb particles showed 4 characteristic peaks as described in Example 3. Mucin-adhered BaCCb particles demonstrated 9 distinctive peaks. Mucin characteristic peaks at 3279cm ¹ , 1634cm ¹ , 1549cm ¹ , 1232cm ¹ , 1115cm ¹ were found respectively at 3384cm ¹ , 1645cm ¹ , 1555cm ¹ , 1213cm ¹ , 1121cm ¹ positions in mucin-adhered BaCCb particles. The presence of characteristic peaks for BaCCb particles and mucin indicates positive adhesion of BaCC> ₃ particles to mucin.

Mucin adhesion to the particles was further assessed by spectrophotometric protein quantification. The percentage of mucin adhesion to BaSC> ₄ , BaSCb and BaCCb particles was calculated by measuring the protein content (mucin) precipitated out with the particles after centrifugation. Protein quantification was done by the Bradford method. The standard curve was prepared with a series of known amount of mucin versus respective absorbance values.

Figure 12 shows the Bradford assay data for mucin adhesion, where (a) shows the standard curve prepared from acetate buffer solution (ABS) versus a known amount of mucin, whereas (b) reveals the percentage of adhesion to BaSCb, BaSCb and BaCCb particles. The BaSCb particles showed the highest mucin adhesion (100%), followed by BaSCb and BaCCb particles with 60 to 70% of mucin adhesion.

Example 7: Oral administration of insulin-loaded BaS0 ₄ , BaS0 ₃ and BaC0 ₃ particles to diabetic animals and management of hyperglycemia

This example illustrates the effect of orally administered protein (insulin) loaded BaSCb or BaSCb and BaCCb nano-precipitates on hyperglycemia.

Induction of diabetes in rats with streptozotocin (STZ)

8 to 12 weeks-old male healthy Wister Kyoto rats (WKY) were subjected to intraperitoneal (IP) injection of STZ (65 mg/kg). A fraction of the rats developed hyperglycemia with persistent high level of peripheral blood glucose (>13mM) within a week or two. Glucose levels in the blood (collected via tail vein) were measured using a glucometer (Terumo, Japan). The rats that did not develop diabetes with the first injection were given a second IP dose of 65 mg/kg STZ.

Effect of orally administrated insulin-loaded BaS04, BaS03 and BaC03 particles on hyperglycaemia Short acting human insulin analogue, Insulin Aspart (NovoRapid, Novonordisk)- loaded BaSC>4, BaSCb and BaC03 particles were administrated to STZ-induced diabetic male Wister Kyoto (WKY) rats. For oral administration, particles were prepared with a high concentration of reactant salts (20X higher than that used in original formulations - Table 2) were loaded with a high dose of insulin Aspart (100 IU/kg).

Rats showing a clear sign of diabetes (peripheral blood glucose level of >13mM) were divided into different control and treatment groups. Each group consists of 3 animals. After taking a baseline blood glucose reading, each rat was given Insulin Aspart-loaded BaSC>4, BaSCb and BaCCb formulations via oral gavage. Rats from the control group were kept untreated (negative control), whereas rats from the positive control group received a solution containing free insulin (100 IU/kg). Treatment groups received 500 pi of solution containing insulin-loaded BaSC> ₄ , BaSCb and BaCC particles by oral gavage. Blood glucose was read at regular time intervals (0.5 hour, 1 hour, 2 hour, 3 hour, and 4 hour). Blood glucose level at "0" hour is the baseline blood glucose reading taken right before oral delivery of insulin-loaded BaSC> ₄ , BaSC and BaCCb particles. The percentage of reduction at any given point of time was calculated from the baseline blood glucose level using the formula below.

Figure 13 shows the data from control groups, i.e. no treatment (negative control) and oral treatment of free insulin (100 IU/kg) (positive control). As expected, none of the groups showed any significant reduction of blood glucose level (p<0.05) at any point in time during the 4 hour study.

Figure 14 shows data from 3 different treatment groups of diabetic rats which were orally given Insulin Aspart-loaded particles of BaSCb, BaSCb and BaCCb. Baseline blood glucose level was read before the treatment. All 3 insulin-loaded Ba salt formulations showed reduction in blood glucose level within 1 hour of oral administration.

For insulin-loaded BaSC> ₄ particles, blood glucose level was significantly low compared to baseline level in 1 to 3 hours (p<0.05). At the 4 ^th hour, the level was still low compared to the baseline level but not significant, with an ascending trend.

Insulin-loaded BaCCb particles showed a significant decrease of blood glucose level at 1 to 2 hours, although blood glucose level started to show a slight rise at the 3 ^rd hour, and by the 4 ^th hour it was back to the baseline level.

Insulin-loaded BaSCh particles showed a reduction in blood glucose levels but the reduction was not significant throughout the 4 hour period.

The P value was calculated by applying one-way ANOVA to confirm any significant reduction of blood glucose level compared to pre-treatment base line blood glucose level, at any point in time after treatment.

Effect of pre-dosing with bicarbonate before oral administration of insulin- loaded BaS04, BaS03 and BaC03 particles on hyperglycaemia

200 mg of bicarbonate was orally administered to each treatment group of diabetic rats followed by oral gavage of insulin-loaded BaSC> ₄ , BaSCh and BaCCh particles, as above. Figure 15 shows the effect on blood glucose levels of pre-administering bicarbonate before oral gavage of insulin-loaded BaSC> ₄ , BaSCh or BaCCh particles, over a 4-hour period.

Figure 16 shows the effect on blood glucose levels of oral administration (a) only with insulin, (b) only with insulin-loaded BaSC> ₄ particles, and (c) with bicarbonate followed by insulin-loaded BaSC> ₄ particles.

Figure 16 clearly shows that the best protocol for reducing blood glucose levels with oral insulin-loaded BaSC> ₄ particles is with pre-treatment oral administration of bicarbonate. Example 8: Albumin loading efficiency of BaS0 ₄ , BaS0 ₃ and BaC0 ₃ particles and subsequent release of albumin from the particles at acidic pH

The capability of the BaSC> ₄ , BaSC and BaCC particles in protecting protein molecules was further assessed using albumin protein against different pHs and enzymatic effect.

Different amounts of albumin bound to the BaSC> ₄ , BaSC and BaCC particles were separated from the unbound proteins by centrifugation and ran on SDS-PAGE to see the band intensity of the bound proteins following Coomassie dye staining (Figure 17). The particles were synthesized in the presence of albumin at pH 7.8. A few assays were done with albumin-loaded particles following exposure of the particles to harsh acidic pH of 1.8. All of the BaSC> ₄ , BaSC and BaCC particles were prepared in the presence of lOOOpg/ml, 800pg/ml and 500pg/ml of albumin at pH 7.8 and the band intensity for the loaded albumin was the same. The albumin band for concentration of lOOpg/ml initially added to prepare the albumin-loaded particles showed slightly lower intensity. That indicates that albumin at 500 pg/ml might saturate the (albumin) binding sites of the particle.

At acidic pH, albumin loaded into BaSC> ₄ particles showed slightly decreased intensity compared to the synthesis pH of 7.8 with clear and intact band. That indicates that the BaSC> ₄ particle can protect albumin from enzymatic degradation even at very harsh acidic pH. However, albumin loaded to BaSCh and BaCCh particles showed significant degradation at harsh acidic pH. For both BaSCh and BaCCh particles, an intact but very low intensity band was found for the highest concentration of 1000 pg/ml of albumin. Bands in lower concentrations of albumin were either too dim in intensity or invisible, indicating a significant amount of protein loss even in particle bound form.

Example 9: Protection of albumin-loaded BaS04, BaS0 ₃ and BaC03 particles from enzymatic (trypsin) digestion

To assess the potential ability of BaSC> ₄ , BaSCh and BaCCh particles to protect particle-bound proteins from enzymatic digestion in the gastrointestinal tract, albumin-loaded BaSC> ₄ , BaSC and BaCCb particles were exposed to trypLE (trypsin mimicking enzyme)-added medium prepared with different pHs (pH 7.4, 6.8 and

1.8). Free undigested and trypsin -digested albumin samples were taken as control.

The trypsin-digested free albumin showed partial fragmentation, whereas albumin proteins loaded into BaSC> ₄ and BaSCh particles remained intact at all 3 different pHs (Figure 18). BaCC particle-bound albumin was found intact in alkaline pH (7.8 and

6.8); however, the protein band was not visible at acidic pH of 1.8 (Figure 18).

Figure 19 shows comparative graphs with relative percentage of protein degradation for different BaSC> ₄ , BaSCb and BaCCb particles. The band intensity was measured using Image! Relative degradation percentage was calculated from the difference in band intensity between trypLE-digested albumin at pH 7.4 and trypLE-digested albumin at subsequent pHs.

Example 10: Protection of albumin-loaded BaS0 ₄ , BaS0 ₃ and BaC0 ₃ particles in simulated gastric fluid (sGF)

Albumin-loaded BaSC!, BaSC and BaCC particles were exposed to simulated gastric fluid (sGF). sGF was prepared with added pepsin and in 3 different pHs 5.0, 2.5 and 1, mimicking the fluctuating pH environment of the stomach. The effect observed on band intensity actually reflects the combined effect of enzymatic activity of pepsin and pH. Figure 20A shows albumin bands from free albumin without any treatment, followed by 3 rows with free albumin exposed to sGF. No visible band for albumin was found when free albumin was exposed to sGF, whereas the 3 visible bands on the gel were from pepsin itself.

Albumin when loaded into the BaSC> ₄ particle and exposed to sGF with different pHs showed a reduction in band intensity with more acidic pH. Both albumin and pepsin bands were visible at pH 5.0 and pH 2.5. While pepsin band intensity remained the same, albumin band intensity seemed to be dimmed at pH 2.5 compared to pH 5. There was no visible band for albumin or pepsin at pH 1.0. As shown in Figure 20B, albumin loaded into BaSC particles and subsequently exposed to sGF with pH 5.0 gave a clear band, whereas loaded albumin exposed to sGF with pH 2.5 showed a decrease in band intensity, and no albumin band was visible at pH 1.0.

Figure 20C demonstrated that the band intensity of the albumin loaded into BaCCh particles remained the same when exposed to sGF with pH 5 and pH 2.5, while no visible band was found at pH 1.0.

The above results suggest that unlike free albumin, salt nanoparticle-bound albumin could better tolerate extreme acidic pH. The lack of visibility of albumin band at pH 1 could be due to overexposure of the insulin-loaded nanoparticles to the extreme acidic pH or non-release of insulin from undegraded particles.

Example 11: Effect of oral administration of insulin-bound SrS03 on blood glucose levels

This example illustrates the effect of orally administered insulin-loaded SrSCh nanoprecipitate on blood glucose levels.

SrSC>3-insulin conjugation was prepared by adding 100 pi of SrCh tlM), 200 IU/kg of insulin and 40 mI of Na ₂ SC> ₃ as per the protocol of Examples 1 and 2. After a 14-hour starvation, 200 mg of bicarbonate was administered followed by oral gavage of SrSC>3-insulin conjugation. Figure 21 shows the effect on blood glucose levels over a 6-hour period.

The above was repeated for SrSC>3-insulin conjugation with 100 IU/kg of insulin and the same treatment protocol was applied after a 14-hour starvation. Figure 22 shows the effect on blood glucose levels over a 6-hour period.

The above was repeated for SrSCh-insulin conjugation with 50 IU/kg of insulin and the same treatment protocol was applied after a 14-hour starvation. Figure 23 shows the effect on blood glucose levels over a 6-hour period. The above was repeated for SrSC>3-insulin conjugation with 100 IU/kg of insulin. 200 mg of sodium bicarbonate was orally administered three times at 10-minute intervals without starvation followed by oral administration of SrSC>3-insulin conjugation with 100 IU/kg of insulin. Figure 24 shows the effect on blood glucose levels over a 6- hour period.

The above was repeated for SrSC -insulin conjugation with 100 IU/kg of insulin, which was orally administered without sodium bicarbonate, after 14-hour starvation. Figure 25 shows the effect on blood glucose levels over a 6-hour period.

As a negative control, free insulin (5 IU/kg) was intravenously administered. Figure 26 shows the effect on blood glucose levels over a 6-hour period.

As could be seen from Figures 21 to 25, there was a significant decrease in blood glucose levels in the beginning. In particular, from Figure 23, it was demonstrated that only a very low concentration of insulin (5 IU /kg) bound to SrSC> ₃ could exert an efficacious effect on reduction of blood glucose levels.

Also pertinent, is the comparison between Figures 22 and 25 i.e. administration of oral bicarbonate before oral gavage with the SrSC>3-insulin conjugation versus nonadministration of bicarbonate before oral gavage with the SrSCh-insulin conjugation.

It is clear from Figures 22 and 25 that oral administration of bicarbonate before oral treatment with insulin-loaded nano-precipitates of this invention significantly increased the percentage of reduction of blood glucose levels in the test subjects.

Example 12: Effect of transferrin and casein surface modification of insulin-loaded BaS04 on blood glucose levels

This example illustrates the effect of transferrin and casein surface modification of insulin-loaded BaSC> ₄ precipitates on blood glucose levels.

BaSCVinsulin loaded precipitates were prepared by adding 100 pi of BaCh tlM), 100 IU/kg of insulin and 40 pi of Na ₂ SC> ₄ , as per the protocol of Examples 1 and 2. The insulin-loaded precipitates were surface modified with addition of 250 pi (lOmg/ml) of transferrin. The transferrin-modified BaSCTHnsulin loaded precipitates were incubated at 37°C for 10 minutes before administration to the rats by oral gavage. Figure 27 shows the effect on blood glucose levels over a 6-hour period following oral administration. Surface modification of the BaSC> ₄ particles with transferrin helped sustained reduction of blood glucose levels, compared to unmodified BaSC> ₄ particles. This could be due to the effect of transferrin in facilitating blood absorption of the insulin-loaded nanoparticles across the intestinal epithelium. For casein-modification, BaSCTHnsulin loaded precipitates were prepared by adding 100 mI of BaCh (1M), 100 IU/kg of insulin and 40 mI of Na ₂ SC> ₄ , as per the protocol of Examples 1 and 2. The insulin-loaded precipitates were surface modified with addition of 250 mI from 1 mg/ml of casein solution. The casein-modified BaSCTr insulin loaded precipitates were incubated at 37°C for 15 minutes and then topped up to a total volume of 500 mI with milliQF O before being administered to the rats by oral gavage. Figures 28 and 29 show the effect on blood glucose levels and the percentage of reduction in blood glucose over a 4-hour period following oral administration. The result suggests that casein, when adsorbed on the surface of the insulin-loaded nanoparticles, could play a positive role in further reducing blood glucose levels, probably by facilitating intestinal transport and blood absorption of the insulin-loaded nanoparticles.

Conclusion

For efficacious oral delivery of protein therapeutics such as insulin, it is necessary to attach the insulin molecule to a "carrier" that carries it safely through the gastrointestinal tract. The "carrier" has to meet the criteria for an oral delivery candidate i.e. able to resist the harsh environment of the gastrointestinal tract and enable the insulin molecules to be absorbed through the intestinal wall. The gastrointestinal tract is a large and complex organ system where each different chamber has its own pH and enzymatic environment. Orally administrated protein therapeutics often show poor bioavailability due to the physical as well as physiological barriers encountered in the gastrointestinal tract. One of the biggest challenges is protein degradation by the extreme acidic pH of the stomach. The second major issue is the enzymatic degradation that occurs in both the stomach and intestine. Even after successfully bypassing the stomach pH and enzymatic action, the protein-loaded particles or the released proteins need to overcome the mucin barrier of the gastrointestinal lining, prior to crossing the epithelium either via transcellular or paracellular route to reach blood circulation.

The particle stability study carried out in Example 4 was to assess the ability of the salt nano-precipitates of this invention to withstand the harsh pH conditions of the gastrointestinal tract, specifically the stomach. Successful oral delivery can only take place if the salt nano-precipitates are resistant even in the face of the fluctuating pH environment of the stomach. Another important factor in oral delivery of therapeutics is stomach and intestinal residence time. Depending on the particle properties, the residence time inside the gut could vary from 20 minutes to 3 hours

(Gao Y. et. al. (2017) ²³ ). An ideal oral delivery carrier must be resistant to both basic and acidic pHs within that timeframe. The stomach has fluctuating pH, which can vary from pH 1.7 to 4.7, whereas intestinal pH is around 6 to 8 (Koziolek M et. al. (2015) ²⁴ ).

Turbidity, which was measured as absorbance at 320nm increases as particle formation is accelerated and decreases as particle formation is inhibited or particle dissolution takes place. The BaSC> ₄ , BaSCh and BaCC>3 precipitates were prepared at pH 7.8 and then exposed to lower pHs. The results showed that BaSC> ₄ particles had the highest synthesis rate at pH 7.8 as well as the best resistance at all pHs throughout 3 hours, followed by the BaCCb and BaSCb particles. All of the BaSC> ₄ , BaSC and BaCCb are suitable as oral delivery carriers based on pH resistance.

Presence of the cation-providing and an ion -providing salts in the nano-precipitate at a sufficiently high concentration to enable adequate saturation of cationic and anionic salts, per volume of the nano-precipitate, is key to strong binding of the target protein molecule and buffering against premature release at an acidic pH.

In Example 5, fluorometric assay was performed with a fixed amount of FUC-insulin added prior to formation of insulin-loaded particles to assess the insulin loading efficiency of BaSC> ₄ , BaSCb and BaCCb particles and subsequent release of insulin from the particles at lower pHs. All of the BaSC> ₄ , BaSCb and BaCCb particles showed very good insulin loading efficiency (80 to 100%). The insulin-loaded particles were also found to be stable as the pH was gradually reduced from 7.78 to 5.0, with almost no release of insulin from the complexes. At pH <5, different degrees of insulin release were observed. Insulin release from BaSCb particles was only at 30% even at very harsh acidic pH, implying that this particular nano-insulin formulation would be stable inside the stomach regardless of whether the subject is fed or in a fasting state. Insulin-loaded BaSCb and BaCCb particles showed almost 80 to 100% release in acidic pH of nearly 1.0.

Mucin of the gastrointestinal lining is the primary barrier for any molecule to cross the intestinal lining and reach systemic circulation. Orally delivered insulin-loaded nano-precipitates of this invention will be in contact with mucin while crossing the intestinal lining.

The adhesion of insulin-loaded BaS0 ₄ , BaS0 ₃ and BaC0 ₃ particles to mucin was assessed with FT-IR and Bradford protein assay kit in Example 6 to predict whether the particles would be capable of crossing the intestinal lining. The FT-IR bands showed characteristic peaks for the BaS0 ₄ , BaS0 ₃ and BaC0 ₃ particles and mucin, and the protein assay quantitatively confirmed mucin adhesion to the particles. All of the BaS0 ₄ , BaS0 ₃ and BaCCb particles were found to have high mucin adhesion (60 to 100%). The effect of orally administrated insulin-loaded nano-precipitates of this invention on hyperglycaemia were assessed in Example 7. All of the insulin-loaded BaSC> ₄ , BaSCb and BaCCb particles resulted in a significant reduction of blood glucose level. It was surprisingly observed that the effect generated by oral administration of the insulin-loaded BaSC> ₄ , BaSCb and BaCCb particles was similar to the effect of subcutaneous delivery of commercial human insulin aspart (Novorapid, Novonordisk) i.e. lasting 4 to 5 hours with a maximum drop in blood glucose level during the 2 to 3 hour mark. For the orally administered insulin-loaded BaSCb, BaSCb and BaCCb particles, onset of action on hyperglycemia started at the 1-hour mark. This could be due to the time required for the insulin-loaded particles to transport the loaded or released insulin into the blood stream. In particular, the BaSCb and BaCCb particles were found to dramatically reduce hyperglycaemia, working in the same way as subcutaneously administered insulin aspart in terms of onset and duration of action. The percentage of reduction of blood glucose level in comparison to the baseline level showed a maximum reduction below 50% at any point in time for all BaSCb, BaSCb and BaCCb particles.

The results of Example 11 suggests that SrSCb particles could also help in protecting insulin from degradation by acidic pH and hydrolytic enzymes in the gastrointestinal (GIT) tract while enabling blood absorption of insulin across the intestinal epithelium.

As will be readily apparent to those skilled in the art, the present invention may easily be produced in other specific forms without departing from its scope or essential characteristics. The present embodiments are, therefore, to be considered as merely illustrative and not restrictive, the scope of the invention being indicated by the claims rather than the foregoing description, and all changes which come within therefore intended to be embraced therein. References

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