Oral formulation and method of preparing thereof

Abstract

The invention provides an oral formulation and the method of preparation thereof for the delivery of a bioactive agent comprising a bioactive agent, a pH modifier, and a solid lipid matrix material, wherein the oral formulation is in the form of solid lipid microparticles formed from the solid lipid matrix material that encapsulates the bioactive agent and the pH modifier. In one embodiment, the bioactive agent is selected from one or more of glucagon-like peptide- 1 (GLP-1) analogues, luteinizing hormone-releasing hormone (LHRH) and gonadotropin-releasing hormone (GnRH), the pH modifier is a weak acid selected from one or more of phosphoric acid, citric acid, lactic acid and malic acid, and the solid lipid matrix material is selected from one or more of glyceryl behenate, glyceryl palmitostearate, tristearin, trimyristin, trilaurin, and stearic acid.

Description

[0001] ORAL FORMULATION AND METHOD OF PREPARING THEREOF

[0002] FIELD OF INVENTION

[0003] The present invention provides oral formulations for the delivery of a bioactive agent and methods of preparing the oral formulations.

[0004] BACKGROUND

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] In accordance with a study by the United Nations, the global population is set to reach an estimated 9.7 billion by 2050. This projected population growth underscores an imminent increase in the demand for food, inclusive of aquaculture products. A report by the Blue Food Assessment suggests that the global consumption of aquatic food products will likely double by 2050, relative to 2015 levels. With natural resources such as wild-caught fish potentially being insufficient to meet this surge in demand, there is a pressing need to enhance the productivity of farmed fish.

[0007] Crucial to optimizing fish farming is the frequency of spawning. By enhancing the rate of spawning, the escalating demand for aquaculture products can be met. The artificial induction of spawning via hormonal stimulation plays a pivotal role in both increasing the yield of farmed fish and in the conservation of threatened and endangered species. It facilitates sexual maturation and spawning. In farmed fish, environmental factors such as tank size, feed availability, and stress levels can impede natural spawning, particularly in certain species like mackerels and tunas, which reportedly require hormonal treatment for successful spawning.

[0008] The current mainstream method for inducing hormone-driven spawning relies heavily on manual injection. This labour-intensive process carries inherent risks such as disruption of spawning efficiency and high levels of stress to the fish. Commonly used synthetic hormones include domperidone (DOM), human chorionic gonadotropin, and luteinizing hormone- releasing hormone (LHRH). These are prepared in syringes and injected intramuscularly into the fish. This method can induce significant stress, potentially leading to secondary infections and even mortality. Moreover, the injection process and handling can cause stress in broodstock, triggering physiological changes that can result in poor spawning performance. A promising alternative lies in the oral delivery of hormones to induce fish spawning, a method which eliminates the need for manual injection. This non-invasive approach promises several advantages, including reduced handling stress, greater convenience, and potentially enhanced efficiency.

[0009] Despite previous studies demonstrating successful spawning using oral hormone delivery, the method requires exceptionally high dosages due to the hormone's low bioavailability, thus hindering the commercial development of oral hormone products (McLean E, Parker DB, Warby CM, Sherwood NM, Donaldson EM. Gonadotropin release following oral delivery of luteinizing hormone-releasing hormone and its superactive analogue (des-Gly10[D-Ala6] LHRH ethylamide) to 17p-oestradiol-primed coho salmon, Oncorhynchus kisutch (Walbaum). Journal of Fish Biology. 1991 ;38(6):851 -858. doi:10.111 1 / j.1095-8649.1991 .tb03625.x; Thomas P, Boyd NW. Dietary administration of an LHRH analogue induces spawning of spotted seatrout (Cynoscion nebulosus'). Aquaculture. 1989;80(3-4):363-370. doi:10.1016 / 0044-8486(89)90183-X). These challenges inspire the development of an oral delivery encapsulation system for the delivery of gastrointestinal environment labile bioactive agents like hormone, ensuring protection and absorption. This approach can improve spawning efficiency and increase the rate of successful spawning in a safe and efficient manner.

[0010] Thus, there is a need for alternative and / or improved oral formulations for the delivery of a bioactive agent (including hormones), and methods of preparing such oral formulations.

[0011] SUMMARY

[0012] Aspects and embodiments of the current invention will now be described by reference to the following numbered clauses.

[0013] 1 . An oral formulation for the delivery of a bioactive agent, the formulation comprising: a bioactive agent; a pH modifier; and a solid lipid matrix material, wherein: the oral formulation is in the form of solid lipid microparticles formed from the solid lipid matrix material that encapsulates the bioactive agent and the pH modifier.

[0014] 2. The oral formulation according to Clause 1 , wherein the pH modifier is selected to lower the pH, so as to protect the bioactive agent from gastrointestinal enzymes at its point of release from the oral formulation. 3. The oral formulation according to Clause 1 or Clause 2, wherein the solid lipid matrix material is selected from one or more of the group consisting of a triglyceride, a fatty acid, and waxes.

[0015] 4. The oral formulation according to Clause 3, wherein the solid lipid matrix material is selected from one or more of the group consisting of glyceryl behenate, glyceryl palmitostearate, tristearin, more particularly, trimyristin, trilaurin, and stearic acid, optionally wherein the solid lipid matrix material is trimyristin.

[0016] 5. The oral formulation according to any one of the preceding clauses, wherein the bioactive agent is an active ingredient that is sensitive to enzymes within the gastrointestinal tract.

[0017] 6. The oral formulation according to Clause 5, wherein the bioactive agent is selected from one or more of a drug, a vaccine, a supplement, more particularly, a protein and a peptide

[0018] 7. The oral formulation according to Clause 6, wherein the bioactive agent is selected from one or more of a hormone, a vaccine, and a peptide drug.

[0019] 8. The oral formulation according to Clause 7, wherein the bioactive agent is selected from one or more of glucagon-like peptide-1 (GLP-1 ) analogues, luteinizing hormone- releasing hormone (LHRH) and gonadotropin-releasing hormone (GnRH), optionally wherein the bioactive agent is luteinizing hormone-releasing hormone (LHRH).

[0020] 9. The oral formulation according to any one of the preceding clauses, wherein the pH modifier is a weak acid, optionally wherein the weak acid has a pKa of from 2 to 5.

[0021] 10. The oral formulation according to Clause 9, wherein the weak acid is selected from a group consisting of one or more of phosphoric acid, citric acid, lactic acid and malic acid, optionally wherein the weak acid is citric acid.

[0022] 11 . The oral formulation according to any one of the preceding clauses, wherein the oral formulation further comprises a release enhancing agent, optionally wherein the release enhancing agent is a liquid lipid material, further optionally wherein the liquid lipid material is coconut oil.

[0023] 12. The oral formulation according to any one of the preceding clauses, wherein the solid lipid matrix material is trimyristin; the bioactive agent is luteinizing hormone-releasing hormone (LHRH); and the pH modifier is citric acid; optionally wherein the oral formulation further comprises a release enhancing agent, wherein the release enhancing agent is coconut oil.

[0024] 13. The oral formulation according to Clause 11 or Clause 12, wherein the mass ratio of the solid lipid matrix material to the liquid lipid material, when present, is from 2:1 to 20:1 (e.g., about 7:3), such as from 3:1 to 10:1 (e.g., about 4:1), optionally wherein the mass ratio of the solid lipid material and the liquid lipid material is about 9:1 .

[0025] 14. The oral formulation according to any one of the preceding clauses, wherein the pH modifier is present in the solid lipid microparticle in an amount of from 1wt% to 20 wt%, such as about 5wt% to 15 wt%, such as 7.5 wt% to 12.5 wt% based on the mass of the solid lipid microparticle, optionally wherein the pH modifier is present in the solid lipid microparticle in an amount of about 1 1 .61wt% based on the mass of the solid lipid microparticle.

[0026] 15. The oral formulation according to any one of the preceding clauses, wherein the bioactive agent is present in the solid lipid microparticle in an amount of from 0.01 wt% to 1 wt% based on the mass of the solid lipid microparticle, optionally wherein the bioactive agent is present in in the solid lipid microparticle an amount of about 0.0127 wt% based on the mass of the solid lipid microparticle.

[0027] 16. The oral formulation according to any one of the preceding clauses, wherein each microparticle has a diameter of from 50 pm to 750 pm, such as from 100 to 500 pm, as measured by scanning electron microscopy (SEM).

[0028] 17. A method of preparing an oral formulation according to any one of Clauses 1 to 16, the method comprising the steps of:

[0029] (i) providing a powder comprising a bioactive agent and a pH modifier, a heated lipid matrix material in a liquid state, where the lipid matrix material is a solid lipid matrix material at room temperature, an emulsifier solution; and

[0030] (ii) mixing the powder with the heated lipid matrix material to form a mixture that was then contacted with the emulsifier solution to provide the oral formulation. 18. The method according to Clause 17, wherein the lipid matrix material in step (I) is heated to a temperature of from 60°C to 70°C, optionally wherein the lipid matrix material in step (I) is heated to a temperature of about 65°C.

[0031] 19. The method according to Clause 17 or Clause 18, wherein the powder comprising the bioactive agent and the pH modifier in step (i) is obtained from subjecting the bioactive agent and the pH modifier to a milling step.

[0032] 20. The method according to any one of Clauses 17 to 19, wherein step (i) further comprises providing a release enhancing agent, and step (ii) further comprises mixing the powder with the heated lipid matrix material and the release enhancing agent to form a mixture that was then contacted with the emulsifier solution to provide the oral formulation. optionally wherein the release enhancing agent is a liquid lipid material.

[0033] 21 . The method according to Clause 20, wherein the mass ratio of the heated lipid matrix material to the liquid lipid material, when present, is from 2: 1 to 20: 1 (e.g., about 7:3), such as from 3:1 to 10:1 (e.g., about 4:1), optionally wherein the mass ratio of the solid lipid material and the liquid lipid material, when present, is about 9:1 .

[0034] 22. The method according to any one of Clause 17 to 21 , wherein the mass ratio of the bioactive agent to the pH modifier in the powder in step (i) is from 1 :500 to 1 :1500, such as 1 :750 to 1 :1250, optionally wherein the mass ratio of the bioactive agent and the pH modifier in the powder in step (i) is about 1 :1000.

[0035] 23. The method according to any one of Clauses 17 to 22, wherein the mass ratio of the powder to the heated lipid matrix material and the liquid lipid material, when present, in step (ii) is from 1 :20 to 7:10, such as about 1 :10, such as about 1 :5, such as about 3:10, such as about 2:5, such as about 1 :2, such as about 3:5, optionally wherein the mass ratio of the powder to the heated lipid matrix material and the liquid lipid material, when present, in step (ii) is about 1 :2.

[0036] 24. The method according to any one of Clauses 17 to 23, wherein one or both of the following apply:

[0037] (a) the mass ratio of the pH modifier to the heated lipid matrix material and the liquid lipid material, when present, in step (ii), is from 1 :1 to 1 :5, such as about 1 :1 , about 1 :2, about 1 :3, about 1 :4 and about 1 :5, optionally wherein the mass ratio of the pH modifier and the heated lipid matrix material and the liquid lipid material, when present, in step (ii), is about 1 :2. (b) the mass ratio of the bioactive agent to the heated lipid matrix material and the liquid lipid material, when present, in step (ii), is from 1 :1000 to 1 :3000, such as about 1 :1500, about 1 :2000, about 1 :2500, optionally wherein the mass ratio of the bioactive agent to the heated lipid matrix material and the liquid lipid material, when present, in step (ii) is about 1 :2000.

[0038] 25. The method according to any one of Clauses 17 to 24, wherein one or both of the following apply:

[0039] (a) the pH modifier is encapsulated at an efficiency of from 25% to 50%, such as about 35%; and

[0040] (a) the bioactive agent is encapsulated at an efficiency of from 25% to 50%, such as about 38.3%.

[0041] 26. A fish feed composition comprising an oral formulation according to any one of Clauses 1 to 16.

[0042] 27. Use of the oral formulation according to Clauses to 1 to 16 or the fish feed composition according to Clause 26 for aquaculture of a fish.

[0043] 28. The use according to the Clause 27, wherein the bioactive agent is a hormone, and the hormone is to be administered at a dosage of from 10 pg / kg to 50 pg / kg based on the mass of the fish, such as about 20 pg / kg or 40 pg / kg ,

[0044] BRIEF DESCRIPTION OF DRAWINGS

[0045] FIG. 1 depicts the digital image (a) and SEM image (b) of the trimyristin SLMs encapsulating citric acid. Cross-sectional image (c) shows citric acid loaded into the lipid matrix of trimyristin. Protective effect of the different concentrations of the citric acid in a simulated intestinal environment (d), release of citric acid from SLMs of trimyristin (D114) only and trimyristin with coconut oil (mass ratio of 9:1) as additive in simulated intestinal environment (e) and release of hormone LHRHa from SLMs of trimyristin with coconut oil (mass ratio of 9:1 ) in simulated intestinal environment (f).

[0046] FIG. 2 depicts the stability of the encapsulated LHRHa hormone microparticles (trimyristin SLM without coconut oil) studied in water environment and simulated gastric fluid (SGF) for a period of 2 hours. FIG. 3 depicts the 11 -KT concentration in plasma after single force-feeding of the encapsulated LHRHa hormone microparticles (trimyristin SLM with coconut oil) compared to injection-based delivery. Two doses (low and high dosage) of the encapsulated hormone were fed. Data presented as an average ± SD of n=3 fish.

[0047] FIG. 4 depicts the 1 1 -KT concentration in fish plasma after force-feeding of the feed incorporating encapsulated LHRHa hormone microparticles (trimyristin SLM with coconut oil) compared with free hormone in feed and injection-based delivery method. The level of 1 1 -KT in the fish with encapsulated hormone is comparable to the injection method. A second feeding of the encapsulated hormone in the encapsulated hormone oral delivery group after 24 hours ensured retention of increased levels of 1 1 -KT in the fish for up to 60 hours. Data presented as an average of n=3 fish.

[0048] FIG. 5 depicts the spawning performance of orally delivered encapsulated LHRHa hormone microparticles as compared to injection of LHRHa in Asian Seabass broodstock: eggs produced in millions (a), fertilization and hatching rate of eggs (b), and larvae survived at Day 1 (c).

[0049] FIG. 6 depicts the comparison of nutrient composition of eggs spawned via injection of LHRHa vs orally delivered encapsulated LHRHa hormone microparticles: fatty acids profile (a,b), and amino acids (c).

[0050] DESCRIPTION

[0051] The present inventors have developed an encapsulation system to ensure the secure delivery of a bioactive agent at the site of absorption in the gastrointestinal tract. The encapsulation system is in the form of solid lipid microparticles formed from a solid lipid matrix material encapsulating a bioactive agent and an acidic modifier. The design of the encapsulation system allows for a targeted and controlled release of the encapsulated contents (i.e., the bioactive agents and the acidic modifier) specifically within the intestinal environment. The solid lipid matrix (SLM) serves as a barrier against environmental factors such as water leaching and gastric degradation, which may otherwise disrupt the encapsulated contents. The acidic modifier regulates the local intestinal pH inhibiting enzymatic activity, thereby protecting the bioactive agent and improving its absorption. As demonstrated in the present Examples, the encapsulation system may be use in an oral formulation for aquaculture to improve fish spawning - the encapsulation system is used to encapsulate fish spawning hormone as the bioactive agent and citric acid as the pH modifier. The oral formulation provides several advantages over conventional means of administering the hormone i.e., via injection, such as reduced handling stress, greater convenience and potentially enhanced efficiency.

[0052] Thus, in a first aspect of the invention, there is provided an oral formulation for the delivery of a bioactive agent, the formulation comprising: a bioactive agent; a pH modifier; and a solid lipid matrix material, wherein: the oral formulation is in the form of solid lipid microparticles formed from the solid lipid matrix material that encapsulates the bioactive agent and the pH modifier.

[0053] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of’ or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0054] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0055] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of two or more such compounds, reference to “a composition” includes mixtures of two or more such compositions, and the like.

[0056] When used herein, the term “pH modifier” refers to a substance that may be used to adjust the pH of the local environment when released from the solid lipid microparticles. In certain embodiments, the pH may be selected to lower the pH, so as to protect the bioactive agent from gastrointestinal enzymes at its point of release from the oral formulation. Without wishing to be bound by theory, the pH modifier may inhibit the activity of the gastrointestinal enzymes, thereby protecting the bioactive agent from gastrointestinal enzymes and improving the absorption the bioactive agent in the gastrointestinal tract.

[0057] In certain embodiments, the pH modifier may be a weak acid. The weak acid may have a pKa of from 2 to 5, such as from 3 to 5, or from 4 to 5. The weak acid may have one or more pKa values below 5. In certain particular embodiments, the weak acid may be selected from a group consisting of one or more of phosphoric acid, citric acid, lactic acid, and malic acid. In certain exemplary embodiments, the weak acid may be citric acid.

[0058] When used herein, the term “solid lipid matrix material” refers to any suitable lipid-based material which is solid at the relevant temperature i.e., room temperature (about 20°C) or body temperature (about 37°C). Advantageously, the microparticle matrix forms a physical barrier between the payload (e.g., the bioactive agent and the pH modifier) and the environment (e.g., gastric fluid) until its intended point of release (e.g., in the presence of lipase at the intestinal region). Furthermore, the lipid matrix material minimises leaching of the bioactive agent into the environment (e.g., water when the oral formulation is added to a fish feed composition used for aquaculture).

[0059] In certain embodiments, the solid lipid matrix material may be selected from one or more of the group consisting of a triglyceride, a fatty acid, and waxes. In certain particular embodiments, the solid lipid matrix material may be selected from one or more of the group consisting of glyceryl behenate, glyceryl palmitostearate, tristearin, more particularly, trimyristin, trilaurin, and stearic acid. In certain exemplary embodiments, the solid lipid matrix material may be trimyristin.

[0060] When used herein, the term “bioactive agent” may refer to any substance that may display a biological activity when taken into the body of an organism. Any suitable bioactive agent may be used. In certain embodiments, the bioactive agent may be an active ingredient that is sensitive to enzymes within the gastrointestinal tract. In certain particular embodiments, the bioactive agent may be selected from one or more of a drug, a vaccine, a supplement, more particularly, a protein and a peptide. In certain more particular embodiments, the bioactive agent may be selected from one or more of a hormone, a vaccine, and a peptide drug. For the avoidance of doubt, more than one bioactive agent can be encapsulated in the solid lipid microparticle.

[0061] As demonstrated in the present Examples, the bioactive agent may be a hormone. In certain embodiments, the bioactive agent maybe selected from one or more of glucagon-like peptide- 1 (GLP-1 ) analogues, luteinizing hormone-releasing hormone (LHRH) and gonadotropinreleasing hormone (GnRH). In certain exemplary embodiments, the bioactive agent may be luteinizing hormone-releasing hormone (LHRH).

[0062] In certain embodiments, the oral formulation may further comprise a release enhancing agent. When used herein, the term “release enhancing agent” refers to any suitable substance that enhances the release of the payload (i.e., the bioactive agent and the pH modifier), meaning the payload may be released at a faster rate and / or in a shorter amount of time. In certain particular embodiments, the release enhancing agent may be a liquid lipid material. When used herein, the term “liquid lipid material” refers to one which is liquid at the relevant temperature i.e., room temperature (about 20°C) or body temperature (about 37°C). In certain exemplary embodiments, the liquid lipid material may be coconut oil.

[0063] In certain exemplary embodiments: the solid lipid matrix material may be trimyristin; the bioactive agent may be luteinizing hormone-releasing hormone (LHRH); and the pH modifier may be citric acid; optionally when the oral formulation further comprises a release enhancing agent, the release enhancing agent may be coconut oil.

[0064] The release enhancing agent may be present in any suitable amount provided that it does not interfere with the formation of the solid lipid microparticles. In certain embodiments, the mass ratio of the solid lipid matrix material to the liquid lipid material, when present, may be from 2:1 to 20:1 (e.g., about 7:3), such as from 3:1 to 10:1 (e.g., about 4:1 ). In certain exemplary embodiments, the mass ratio of the solid lipid material and the liquid lipid material may be about 9:1. Advantageously, adjusting the mass ratio of the solid lipid matrix material and the liquid lipid material affects the morphological properties and release properties of the solid lipid microparticles.

[0065] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, within 1%, within 0.5%, within 0.1 %, within 0.05%, within 0.01 %, within 0.005%, or within 0.001% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0066] The pH modifier may be present in any suitable amount so as to inhibit the activity of the gastrointestinal enzymes at the point of release of the oral formulation. In certain embodiments, the pH modifier may be present in the solid lipid microparticle in an amount of from 1wt% to 20%, such as about 5wt% to 15 wt%, such as 7.5 wt% to 12.5 wt% based on the mass of the solid lipid microparticle. In certain exemplary embodiments, the pH modifier may be present in the solid lipid microparticle in an amount of about 11 .61 wt% based on the mass of the solid lipid microparticle.

[0067] The bioactive agent may be present in any suitable amount. In certain embodiments, the bioactive agent may be present in the solid lipid microparticle in an amount of from 0.01 wt% to 1 wt% based on the mass of the solid lipid microparticle. In certain exemplary embodiments, the bioactive agent may be present in in the solid lipid microparticle an amount of about 0.0127 wt% based on the mass of the solid lipid microparticle.

[0068] In certain embodiments, the solid lipid microparticle may have a diameter of from 50 pm to 750 pm, such as from 100 to 500 pm. The diameter of the solid lipid microparticles may be measured by any conventional techniques and / or device known in the art, such as scanning electron microscopy (SEM).

[0069] In a second aspect of the invention, there is provided a method of preparing an oral formulation as disclosed hereinbefore, the method comprising the steps of:

[0070] (I) providing a powder comprising a bioactive agent and a pH modifier, a heated lipid matrix material in a liquid state, where the lipid matrix material is a solid lipid matrix material at room temperature, an emulsifier solution; and

[0071] (ii) mixing the powder with the heated lipid matrix material to form a mixture that was then contacted with the emulsifier solution to provide the oral formulation.

[0072] As details of the solid lipid matrix material, bioactive agent, and the pH modifier have already been described above, they are omitted here for brevity.

[0073] As will be appreciated, the lipid matrix material is heated above its melting point to a suitable temperature such that the bioactive agent and the pH modifier may be dissolved or dispersed in the liquid melt. In certain embodiments, the lipid matrix material in step (i) may be heated to a temperature of from 60°C to 70°C. In certain exemplary embodiments, the lipid matrix material in step (i) is heated to a temperature of about 65°C. Advantageously, lipid matrix materials with low melting points are preferred to prevent any possible degradation of the bioactive agent and the pH modifier during the preparation process. As will be appreciated, the powder comprising the bioactive agent and the pH modifier may be obtained from any suitable means. In certain embodiments, the powder comprising the bioactive agent and the pH modifier in step (i) may be obtained from subjecting the bioactive agent and the pH modifier to a milling step. Advantageously, providing the bioactive agent and the pH modifier in powder form improves the dissolution / dispersion in the heated lipid matrix material.

[0074] As mentioned above, the oral formulation may comprise a release enhancing agent. As such, in certain embodiments, step (i) may further comprise providing a release enhancing agent, and step (ii) may further comprise mixing the powder with the heated lipid matrix material and the release enhancing agent to form a mixture that was then contacted with the emulsifier solution to provide the oral formulation.

[0075] In certain embodiments, the mass ratio of the heated lipid matrix material to the liquid lipid material, when present, may be from 2: 1 to 20: 1 (e.g., about 7:3), such as from 3:1 to 10:1 (e.g., about 4:1 ). In certain exemplary embodiments, the mass ratio of the solid lipid material and the liquid lipid material, when present, may be about 9:1. Advantageously, adjusting the mass ratio of the solid lipid matrix material and the liquid lipid material affects the morphological properties and release properties of the solid lipid microparticles.

[0076] In certain embodiments, the mass ratio of the bioactive agent to the pH modifier in the powder in step (i) may be from 1 :500 to 1 :1500, such as 1 :750 to 1 :1250. In certain exemplary embodiments, the mass ratio of the bioactive agent and the pH modifier in the powder in step (i) is about 1 :1000. Advantageously, adjusting the mass ratio of the bioactive agent to the pH modifier ensures proper protection of the bioactive agent at the point of release of the oral formulation.

[0077] In certain embodiments, the mass ratio of the powder to the heated lipid matrix material and the liquid lipid material, when present, in step (ii) may be from 1 :20 to 7:10, such as about 1 :10, such as about 1 :5, such as about 3:10, such as about 2:5, such as about 1 :2, such as about 3:5. In certain exemplary embodiments, the mass ratio of the powder to the heated lipid matrix material and the liquid lipid material, when present, in step (ii) may be about 1 :2. Advantageously, adjusting the mass ratio of the powder to the heated lipid matrix material and the liquid lipid material, when present affects the encapsulation efficiencies of the bioactive agent and the pH modifier.

[0078] In certain further embodiments, one or both of the following may apply: (a) the mass ratio of the pH modifier to the heated lipid matrix material and the liquid lipid material, when present, in step (ii), may be from 1 :1 to 1 :5, such as about 1 :1 , about 1 :2, about 1 :3, about 1 :4 and about 1 :5, optionally wherein the mass ratio of the pH modifier and the heated lipid matrix material and the liquid lipid material, when present, in step (ii), may be about 1 :2.

[0079] (b) the mass ratio of the bioactive agent to the heated lipid matrix material and the liquid lipid material, when present, in step (ii), may be from 1 :1000 to 1 :3000, such as about 1 :1500, about 1 :2000, about 1 :2500, optionally wherein the mass ratio of the bioactive agent to the heated lipid matrix material and the liquid lipid material, when present, in step (ii) may be about 1 :2000.

[0080] As demonstrated in the present Examples, the bioactive agent and the pH modifier may be encapsulated at various efficiencies, depending on the amount of the bioactive agent, the pH modifier and the lipid matrix material. As such, in certain embodiments, one or both of the following may apply:

[0081] (a) the pH modifier may be encapsulated at an efficiency of from 25% to 50%, such as about 35%; and

[0082] (a) the bioactive agent may be encapsulated at an efficiency of from 25% to 50%, such as about 38.3%.

[0083] As demonstrated in the present Examples, the present oral formulation may be used as a feed additive for aquaculture. As such, in another aspect of the invention, there is provided a fish feed composition comprising an oral formulation as disclosed herein.

[0084] As demonstrated in the present Examples, the present oral formulation or fish feed composition may find particular utility in aquaculture. As such, in another aspect of the invention, there is provided use of the oral formulation or the fish feed composition as disclosed hereinbefore for aquaculture of a fish.

[0085] The bioactive agent may be administered at a suitable dosage as determined by the skilled person. In certain embodiments, when the bioactive agent is a hormone, the hormone may be be administered at a dosage of from 10 pg / kg to 50 pg / kg based on the mass of the fish, such as about 20 pg / kg or 40 pg / kg,

[0086] Further aspects and embodiments of the invention are described in the following numbered statements. EXAMPLES

[0087] Materials

[0088] Unless otherwise stated, all materials were obtained from commercial sources. The hormone, LHRHa was purchased from Syndel USA. Trimyristin (Dynasan 1 14) was purchased from IOI Oleo GmbH, and Extra Virgin Coconut Oil (Cold Pressed) was obtained from NTUC Fairprice Co-operative Ltd, Singapore. Poly-vinyl Alcohol (PVA) (CAS No. 9002-89-5), Pancreatin (CAS No. 8049-47-6), and citric acid (CAS No. 77-92-9) were purchased from Sigma-Aldrich. The 11 -keto Testosterone ELISA test kit was purchased from Cayman Chemical Company, USA.

[0089] Example 1 : Solid lipid microparticles encapsulating a spawning hormone, LHRHa and citric acid (CA) as an acidic modifier

[0090] Citric acid alone or together with the hormone, LHRHa was encapsulated in the SLMs. For the latter, LHRHa and citric acid were dissolved in an aqueous solution at a ratio of 1 :1000, and freeze-dried to obtain a dry product. The ratio is identified to be suitable for the acidic modifier to perform its intended protective function in a simulated intestinal environment as shown in FIG. 1 (d). The product was then milled to obtain a powder. The bioactive powder was a mixture of the hormone and citric acid.

[0091] Trimyristin or a combination of trimyristin and coconut oil was used to fabricate the microencapsulation system. The lipids were subjected to temperature of 65°C which is higher than the melting point of the lipids. Milled dry powder of the citric acid or the bioactive powder made up of hormone and citric acid was added to form a homogenous mixture at different ratios of milled powder to lipid such as 1 :10, 1 :5, 1 :2, 2:5, 3:5 and 3:10. The optimized ratio of bioactive powder to lipid for high encapsulation efficiency and loading efficiency was 1 :2. A microemulsion was then formed by adding the mixture into an emulsifier solution of 50ml 1% PVA which was also maintained at 65°C. The heated microemulsion was rapidly cooled down by the addition of cold (4°C) 0.1% PVA to form solid lipid particles. The particles thus formed can be extracted by a series of filtration steps using Buchner funnel filtering followed by freeze drying. The SLMs encapsulating the citric acid and the hormone, LHRHa were then subjected to physiochemical characterizations.

[0092] SLMs of 100 to 500pm (n=10) were observed under scanning electron microscopy (SEM) with a spherical shape and a smooth morphology as shown in FIG. 1(b). The cross-sectional view shows the incorporation of the citric acid powder in an SLM (FIG. 1(c)). An optimized ratio of citric acid to trimyristin of 1 :2 yielded an encapsulation efficiency of 35% of citric acid, corresponding to a loading capacity of 11 .61% which was studied by dissolving the particles in organic solvent, extracting the citric acid in water and quantifying using a High-Performance Liquid Chromatography (HPLC). About 34% of the citric acid was released at 3h and 55% at 6h when the SLMs were incubated in simulated intestinal conditions containing enzymes. The addition of coconut oil (10% of the trimyristin) as an additive improved the release of citric acid leading to a 20% increase at 3h i.e. 54% and 72% at 6h as shown in FIG. 1(d). For this formulation, the encapsulation efficiency of the hormone was found to be 38.3% and the loading capacity was 0.0127% (0.127|ig / mg). Hormone release of about 68% within 6 hours in simulated intestinal fluid was observed as shown in FIG. 1(f).

[0093] While the hormone and citric acid are highly soluble in water, the SLMs provided high leaching resistance to hormone when suspended in aqueous solution as shown in FIG. 2. The scenario is crucial in an aquaculture setting where leaching affects the accessibility of nutrients and drugs during oral delivery via incorporation into the feed. Similarly, encapsulation can also improve the stability in the gastric region as shown in FIG. 2. The low pH and the presence of enzymes like pepsin can be detrimental to the hormone. However, the encapsulation of the hormone ensured no losses when incubated in a simulated gastric fluid.

[0094] The SLMs were also then studied for bioactivity via feeding as standalone microparticles or by incorporating them into the fish feeds. Barramundi broodstock fish were used as models for the in vivo studies. All encapsulated samples used in the following in vivo studies contain coconut oil in the trimyristin SLM (mass ratio of trimyristin to coconut oil is 9:1 ).

[0095] When used as standalone microparticles, a low and high dosage of the SLMs loaded with citric acid and hormone (H+CA+SLMs(high dose, 40pg / kg)) and H+CA+SLMs(low dose, 20pg / kg )) were force-fed to the Barramundi fish (n=3 per group) with average weight of 2050g and compared with controls that included hormone injection (H injection), normal control receiving none of the treatment (Normal), citric acid only containing SLMs (CA+En (w / o H)) , hormone (low dose) and citric acid without encapsulation (H+CA (w / o En)). The bioactivity of the delivered hormone is evaluated by the stimulation and production of a sex hormone i.e., 11 -keto testosterone (1 1 -KT), which is a key indicator of fish maturation and spawning. The hormone levels in the injection group peaked at 12h (127.48 pg / ml), while that of the high- dose encapsulated hormone reached a peak of 90.3 pg / ml at 12 has shown in FIG. 3. This provided proof of degradation and absorption of the hormone from the SLMs leading to bioactivity. Similarly, a higher dosage showed higher bioactivity, comparable to the injection mode of delivery. As SLMs are emptied with the gastrointestinal passage of the food, a decline in the levels of 11 -KT was observed in all the oral feeding groups. Overall, a proof of concept was demonstrated using the in vivo oral delivery trial.

[0096] The incorporation of the microparticles into the Asian Seabass, (Lates calcarifer) fish feed, followed by an oral delivery was also investigated. The experimental group i.e., SLMs containing encapsulated hormone and citric acid (Encapsulated Hormone oral delivery) was compared with fish injected with hormone (Hormone Injection), feed incorporating unencapsulated free hormone (Hormone direct feeding) and fish with no feeding or injection (Normal Control). Initial feeding of 20pg / kg of fish followed by an additional feeding 20pg / kg of fish of the encapsulated hormone after 24 hours was also performed as a strategy to match the elevated levels of 11 -KT as observed in FIG. 3 . Results showed that the encapsulated hormone performed significantly better than the free hormone added to the feed as shown in FIG. 4. This is expected as the free hormone is bound to be degraded by the enzymes of the gastrointestinal tract. However, when protected via encapsulation the hormones showed better absorption and bioactivity. The encapsulated hormone maintained a high level of 11 -KT when fed orally and was comparable to that of the injection method. Additional feeding of the hormone at 24 hours retained the elevated levels of the 11 -KT in the encapsulated group. As oral feeding is non-invasive, multiple feedings to obtain better spawning results are still feasible. Overall, the results established encapsulated hormone as an alternative method for conventional injection-based delivery of the hormones like LHRHa.

[0097] The spawning of the Asian Seabass (Lates calcarifer) broodstock (n=20-25, 5kg each) was investigated by orally delivering the encapsulated hormones via fish feeds and compared with injection of hormone. The microparticles were added to the ground commercial fish feeds at high hormone dosage of 40pig / kg of fish and reshaped into fish feeds for feeding the broodstock fish. The spawning of the broodstock post feeding are shown in FIG. 5. Both oral delivery and injection treatment were successful in inducing spawning of eggs. The eggs produced was found to be higher in injection group (18.46 million) as compared to encapsulated oral delivery group (12.95 million). The fertilization rate of the eggs was higher for oral encapsulated hormone at 80.5% as compare to injection group which showed the fertilization rate to be about 51%. Similarly, the hatching rate of the fertilized eggs was also improved for oral encapsulated hormone (88%) as compared to injection group (51%). Finally, the larvae survived at Day 1 was also higher for oral encapsulated hormone group (8.35 million) vs injection (3.8 million). The findings demonstrate that oral delivery of encapsulated spawning hormone is a promising and effective alternative to injections in aquaculture. While the total eggs production was higher for the injection group, the fertilization, hatching and larvae survivability results show that the eggs produced through oral delivery were of superior quality. The superior spawning performance observed in the orally fed groups can therefore be attributed to the absence of handling stress, leading to better broodstock health and improved spawning outcomes. This improvements in viable larvae will translate to higher productivity for the farms.

[0098] The eggs spawned via the oral delivery showed notable improvement in the nutritional composition as shown in Table 1 and FIG. 6. The oral group showed notable increment in fat and protein levels over injection. While both treatments showed similar fatty acids profile, the injection group showed slightly higher levels of long-chain omega-3 fatty acids (EPA, DPA, DHA). However, lower trans fats and higher monounsaturated fats (oleic acid, eicosenoic acid) and omega-7 / 9 fats showed improved overall fatty acids profile for the oral encapsulated hormone group. Additionally, the eggs from the oral group had higher levels of essential amino acids such as threonine, valine, isoleucine, leucine, phenylalanine, histidine, and methionine which are crucial for embryonic tissue growth, protein synthesis, antioxidant protection, and metabolic support. Overall, these nutritional compositions of the eggs suggest that the encapsulated oral hormone delivery led to better nutrient provisioning by the broodstock which might have resulted in improved spawning performance observed in the spawning trials.

[0099] Table 1 : Nutrients composition of eggs spawned via injection and oral delivery of hormone

[0100] Conclusion

[0101] At present there is no encapsulation production in the market for oral delivery of hormones to the fish. Aquaculture is one of the biggest food industries with a market size of around USD 265 billion in 2020. As such, this technology, the first to provide an oral delivery solution for hormones, can have a huge market impact. The advantages of an oral delivery system for these bioactive agents include ease of use via incorporation into the fish feeds, no handling stress to fish and its negative effects on fish health and reducing the need for manpower. Therefore, the product can be a cheaper and safer alternative to the injection method for fish farmers and aquaculture companies around the world. Companies can readily accept the technology for large-scale fabrication which presents an opportunity for broadening commercialization. Specialized feeds incorporating the technology can thus be a new normal for the fish spawning industry.

[0102] The platform technology is also suitable for other bioactive agents like peptides, vaccines and other sensitive products which are impacted by the enzymes of the gastrointestinal tract. Like the aquaculture industry, the injection-based method is still one of the most common techniques for the application of bioactive agents like antimicrobials, vaccines, and other peptides. The technology can be investigated for the oral delivery of these bioactive agents improving patient compliance, treatment efficacy and health. Examples include mitigating patient incompliance by replacing the injection method of delivery of drugs in children with oral delivery, development of orally delivered glucagon-like peptide-1 (GLP-1) analogue as an alternative to injection. Similarly, would be in use of technology to deliver drugs, hormones and vaccines to livestock and pets. Beyond aquaculture, this technology can therefore find its application for both human and animal usage.

[0103] Further aspects and embodiments of the invention are described in the following numbered statements.

[0104] 1. A microparticle comprising: a) A solid lipid matrix; b) A pH modifier; c) A hormone or peptide; and Optionally, d) An additive.

[0105] 2. The microparticle according to Statement 1 , wherein the solid lipid matrix comprises a solid lipid material including triglycerides (such as trimyristin and trilaurin) and / or fatty acids (such as stearic acid).

[0106] 3. The microparticle according to Statement 1 or 2, wherein the pH modifier comprises weak acids such as citric acid, lactic acid and malic acid.

[0107] 4. The microparticle according to Statements 1 to 3, wherein the hormone or peptide comprises glucagon-like peptide-1 (GLP-1 ) or spawning hormones such as LHRHa and SGnRH. 5. The microparticle according to Statements 1 to 4, wherein the additive comprises liquid lipids such as coconut oil.

[0108] Some of the key advantages of this invention are summarized as follows: 1 . Co-delivery of bioactive hormone and acidic modifier in a single encapsulation system,

[0109] 2. High encapsulation and loading efficiency of the hormone resulting in lower dosage of microparticles for achieving the intended functions,

[0110] 3. Protection against leaching of the hormone in the water environment before feeding by the fish and against the stomach’s acidic pH and enzymes after feeding, 4. Targeted and controlled release of the hormone and the acidic modifier in the intestine resulting in sufficient protection and absorption of the hormone for the bioactivity.

Claims

CLAIMS1 . An oral formulation for the delivery of a bioactive agent, the formulation comprising: a bioactive agent; a pH modifier; and a solid lipid matrix material, wherein: the oral formulation is in the form of solid lipid microparticles formed from the solid lipid matrix material that encapsulates the bioactive agent and the pH modifier.

2. The oral formulation according to Claim 1 , wherein the pH modifier is selected to lower the pH, so as to protect the bioactive agent from gastrointestinal enzymes at its point of release from the oral formulation.

3. The oral formulation according to Claim 1 or Claim 2, wherein the solid lipid matrix material is selected from one or more of the group consisting of a triglyceride, a fatty acid, and waxes.

4. The oral formulation according to Claim 3, wherein the solid lipid matrix material is selected from one or more of the group consisting of glyceryl behenate, glyceryl palmitostearate, tristearin, more particularly, trimyristin, trilaurin, and stearic acid, optionally wherein the solid lipid matrix material is trimyristin.

5. The oral formulation according to any one of the preceding claims, wherein the bioactive agent is an active ingredient that is sensitive to enzymes within the gastrointestinal tract.

6. The oral formulation according to Claim 5, wherein the bioactive agent is selected from one or more of a drug, a vaccine, a supplement, more particularly, a protein and a peptide.

7. The oral formulation according to Claim 6, wherein the bioactive agent is selected from one or more of a hormone, a vaccine, and a peptide drug.

8. The oral formulation according to Claim 7, wherein the bioactive agent is selected from one or more of glucagon-like peptide-1 (GLP-1) analogues, luteinizing hormone-releasing hormone (LHRH) and gonadotropin-releasing hormone (GnRH), optionally wherein the bioactive agent is luteinizing hormone-releasing hormone (LHRH).

9. The oral formulation according to any one of the preceding claims, wherein the pH modifier is a weak acid, optionally wherein the weak acid has a pKa of from 2 to 5.

10. The oral formulation according to claim 9, wherein the weak acid is selected from a group consisting of one or more of phosphoric acid, citric acid, lactic acid and malic acid, optionally wherein the weak acid is citric acid.11 . The oral formulation according to any one of the preceding claims, wherein the oral formulation further comprises a release enhancing agent, optionally wherein the release enhancing agent is a liquid lipid material, further optionally wherein the liquid lipid material is coconut oil.

12. The oral formulation according to any one of the preceding claims, wherein the solid lipid matrix material is trimyristin; the bioactive agent is luteinizing hormone-releasing hormone (LHRH); and the pH modifier is citric acid; optionally wherein the oral formulation further comprises a release enhancing agent, wherein the release enhancing agent is coconut oil.

13. The oral formulation according to Claim 11 or Claim 12, wherein the mass ratio of the solid lipid matrix material to the liquid lipid material, when present, is from 2:1 to 20:1 (e.g., about 7:3), such as from 3:1 to 10:1 (e.g., about 4:1), optionally wherein the mass ratio of the solid lipid material and the liquid lipid material is about 9:1 .

14. The oral formulation according to any one of the preceding claims, wherein the pH modifier is present in the solid lipid microparticle in an amount of from 1wt% to 20 wt%, such as about 5wt% to 15 wt%, such as 7.5 wt% to 12.5 wt% based on the mass of the solid lipid microparticle, optionally wherein the pH modifier is present in the solid lipid microparticle in an amount of about 1 1 ,61 t% based on the mass of the solid lipid microparticle.

15. The oral formulation according to any one of the preceding claims, wherein the bioactive agent is present in the solid lipid microparticle in an amount of from 0.01 wt% to 1 wt% based on the mass of the solid lipid microparticle, optionally wherein the bioactive agent is present in in the solid lipid microparticle an amount of about 0.0127 wt% based on the mass of the solid lipid microparticle.

16. The oral formulation according to any one of the preceding claims, wherein each microparticle has a diameter of from 50 pm to 750 pm, such as from 100 to 500 pm, as measured by scanning electron microscopy (SEM).

17. A method of preparing an oral formulation according to any one of Claims 1 to 16, the method comprising the steps of:(i) providing a powder comprising a bioactive agent and a pH modifier, a heated lipid matrix material in a liquid state, where the lipid matrix material is a solid lipid matrix material at room temperature, an emulsifier solution; and(ii) mixing the powder with the heated lipid matrix material to form a mixture that was then contacted with the emulsifier solution to provide the oral formulation.

18. The method according to Claims 17, wherein the lipid matrix material in step (i) is heated to a temperature of from 60°C to 70°C, optionally wherein the lipid matrix material in step (i) is heated to a temperature of about 65°C.

19. The method according to Claim 17 or Claim 18, wherein the powder comprising the bioactive agent and the pH modifier in step (i) is obtained from subjecting the bioactive agent and the pH modifier to a milling step.

20. The method according to any one of Claims 17 to 19, wherein step (i) further comprises providing a release enhancing agent, and step (ii) further comprises mixing the powder with the heated lipid matrix material and the release enhancing agent to form a mixture that was then contacted with the emulsifier solution to provide the oral formulation. optionally wherein the release enhancing agent is a liquid lipid material.21 . The method according to Claim 20, wherein the mass ratio of the heated lipid matrix material to the liquid lipid material, when present, is from 2: 1 to 20: 1 (e.g., about 7:3), such as from 3:1 to 10:1 (e.g., about 4:1 ), optionally wherein the mass ratio of the solid lipid material and the liquid lipid material, when present, is about 9:1 .

22. The method according to any one of Claims 17 to 21 , wherein the mass ratio of the bioactive agent to the pH modifier in the powder in step (i) is from 1 :500 to 1 :1500, such as 1 :750 to 1 :1250, optionally wherein the mass ratio of the bioactive agent and the pH modifier in the powder in step (i) is about 1 :1000.

23. The method according to any one of Claims 17 to 22, wherein the mass ratio of the powder to the heated lipid matrix material and the liquid lipid material, when present, in step (ii) is from 1 :20 to 7:10, such as about 1 :10, such as about 1 :5, such as about 3:10, such as about 2:5, such as about 1 :2, such as about 3:5, optionally wherein the mass ratio of the powder to the heated lipid matrix material and the liquid lipid material, when present, in step (ii) is about 1 :2.

24. The method according to any one of Claims 17 to 23, wherein one or both of the following apply:(a) the mass ratio of the pH modifier to the heated lipid matrix material and the liquid lipid material, when present, in step (ii), is from 1 :1 to 1 :5, such as about 1 :1 , about 1 :2, about 1 :3, about 1 :4 and about 1 :5, optionally wherein the mass ratio of the pH modifier and the heated lipid matrix material and the liquid lipid material, when present, in step (ii), is about 1 :2.(b) the mass ratio of the bioactive agent to the heated lipid matrix material and the liquid lipid material, when present, in step (ii), is from 1 :1000 to 1 :3000, such as about 1 :1500, about 1 :2000, about 1 :2500, optionally wherein the mass ratio of the bioactive agent to the heated lipid matrix material and the liquid lipid material, when present, in step (ii) is about 1 :2000.

25. The method according to any one of Claims 17 to 24, wherein one or both of the following apply:(a) the pH modifier is encapsulated at an efficiency of from 25% to 50%, such as about 35%; and(a) the bioactive agent is encapsulated at an efficiency of from 25% to 50%, such as about 38.3%.

26. A fish feed composition comprising an oral formulation according to any one of Claims 1 to 16.

27. Use of the oral formulation according to Claims to 1 to 16 or the fish feed composition according to Claim 26 for aquaculture of a fish.

28. The use according to the Claim 27, wherein the bioactive agent is a hormone, and the hormone is to be administered at a dosage of from 10 pg / kg to 50 pg / kg based on the mass of the fish, such as about 20 pg / kg or 40 pg / kg,

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