ORAL DELIVERY SYSTEM FOR BIOACTIVE AGENTS
20220409541 · 2022-12-29
Inventors
Cpc classification
A61K9/1652
HUMAN NECESSITIES
A61K9/5036
HUMAN NECESSITIES
A23K50/80
HUMAN NECESSITIES
A61K47/36
HUMAN NECESSITIES
International classification
A61K9/16
HUMAN NECESSITIES
A23K50/80
HUMAN NECESSITIES
A61K47/36
HUMAN NECESSITIES
Abstract
The present invention relates to a functional feed and an oral delivery system for delivery of bioactive macromolecules. The oral delivery system comprises ethylenediammonium alginate which is a vehicle for delivery of macromolecular drugs. The oral delivery system according to the present invention is particularly suitable for use in combination with functional feeds in fish.
Claims
1. A method of therapeutically or prophylactically treating fish, the method comprising: administering to the fish an oral delivery system, the oral delivery system comprising a fish feed pellet comprising ethylenediammonium alginate particles having a mean particle size of no more than 2000 microns, the fish feed pellet comprising an oil suspension wherein the ethylenediammonium alginate particles are vacuum coated thereon, the ethylenediammonium alginate particles comprising ethylenediammonium alginate; and a bioactive agent that is encapsulated or entrapped in the ethylenediammonium alginate; wherein the fish is a coldwater fish that lives in average water temperatures of about 20° C. or lower.
2. The method of claim 1, wherein the fish is an omnivorous or carnivorous species.
3. The method of claim 1, wherein the fish is a gastric fish, a fish with a short intestinal tract, salmonids, basses, breams, codfish, halibut, turbot, flounders, grouper, tuna, tilapia, or pangasius.
4. The method of claim 1, wherein the bioactive agent is selected from the group consisting of proteins, peptides, vaccines, antibodies, antigens, hormones, enzymes, immune stimulants, drugs, probiotics, prebiotics, polynucleotides, nucleotides, and amino acids.
5. The method of claim 1, wherein the mean particle size of the ethylenediammonium alginate particles is no more than 300 μm.
6. The method of claim 1, wherein the particles comprise beads.
7. The method of claim 1, wherein the mean particle size of the ethylenediammonium alginate particles is in the range of 300 μm to 2000 μm.
8. The method of claim 1, wherein the mean particle size of the ethylenediammonium alginate particles is no more than 100 μm.
9. The method of claim 1, wherein the mean particle size of the ethylenediammonium alginate particles is no more than 200 μm.
Description
DESCRIPTION OF THE INVENTION
[0052] Embodiments of the invention will now be described, by the way of examples with reference to the following diagrams, wherein
[0053]
[0054]
[0055]
[0056] The present invention provides a novel oral delivery system for delivery of bioactive agents to the intestine. This oral delivery system is particularly suitable for use in organisms with a short intestinal tract such as carnivorous fish. In particular, the oral delivery system is suitable for oral delivery of bioactive substances in ectothermic animals which can have a digestion at low temperatures.
[0057] In the present invention sodium alginate is cross-linked with ethylenediamine dihydrochloride to form ethylenediammonium alginate. The advantage of this method is that no other chemicals are involved. Furthermore, the described method to cross-link alginate with ethylenediammonium hydrochloride is faster, easier, less expensive, and does not involve dangerous chemicals compared to other known methods.
[0058] In accordance with the present invention, ethylenediammonium alginate is used as an oral delivery system for bioactive agents, in particular in fish. The agents are encapsulated or entrapped in the matrix of ethylenediammonium alginate during the process of cross-linking alginate with ethylenediammonium dications. Preferably, small alginate microbeads are produced during this cross-linking process consisting of two major steps: 1) generating of alginate droplets by devices such as spray or jetting heads fitted with nozzles as those found in aerodynamically assisted jetting or by other methods known to the skilled person for generating droplets of liquids (e.g. aerodynamically assisted jetting, electromagnetic laminar jet breakup, inkjet printing, 3D printing, electro spraying, coaxial air flow induced dripping etc.); 2) collecting alginate droplets in cross-linking solution containing ethylenediammonium. Alter-natively, an emulsion method (e.g., coacervation, internal and external gelation etc.) can be used in combination with ethylenediammonium.
[0059] The ethylenediammonium alginate matrix can typically have the form of any geometric particle shape for particles e.g., fibre, sphere, toroid, ellipsoid and also including fibres and flakes. Preferably, the particles are regular shaped, although said alginate matrix can also be in form of an irregularly shaped particle. In a preferred embodiment said matrix is in form of beads, more preferred in form of spherical beads.
[0060] The resulting alginate beads comprising bioactive agent(s) can be enterally administered either in feed or ex feed (independently, without regard to feed) to target organisms. The preferred mean particle size of the alginate beads is in the range of 1 μm to 10 mm. For associating with feed pellets in a vacuum coater the mean size of alginate beads can typically be in the range of 300 μm and 2000 μm. However, the preferred mean size for pre-extrusional addition is less than 300 μm. Preferred particles sizes are less than 300 μm, more preferable less than 100 μm and most preferable less than 25 μm. The preferred mean size of alginate beads for stand-alone administration (without being in cooperated in a feed) is greater than 100 μm. For this use the preferred size of the beads will typically depend on both, the size of the animal and target dose and may be adjusted correspondingly. However, the preferred route of administration to fish is the oral route with alginate beads in feed.
[0061] The small alginate beads comprising bioactive agent(s) are preferably incorporated into feed pellets by a suitable method such as vacuum infusion coating, before they are orally administered to target organisms, such as a fish. Alternatively, the beads can be mixed with other feed ingredients before pelletizing. These ingredients can for example be conventional feed components known to a skilled person. Preferably, the alginate beads comprising one or several bioactive ingredient(s) are mixed with initial flour (dry mix consisting of all feed ingredients except from oil) prior to extrusion or any other means of making feed pellets.
[0062] A detailed description of a preferred method in agreement with the present invention for cross-linking of alginate with ethylenediamine dihydrochloride as well as a preferred method for production of small ethylenediammonium alginate beads loaded with one or more bioactive agents is provided in the experimental section below. Furthermore, a preferred method according to the present invention for incorporating the alginate beads into feed pellets is provided in said experimental section.
[0063] Experimental section:
[0064] The efficacy of ethylenediammonium alginate (EDA-alginate) was tested and proved in two different experimental settings in vitro and in vivo. Results obtained were compared to a common oral delivery system based on alginate cross-linked with Ca.sup.2+(Ca-alginate).
[0065] As described above, the digestive conditions in fish, being ectothermic and especially in carnivorous fish having a short intestine, differs from those of many other organisms, especially from mammals and humans. Therefore, in the present invention, the oral delivery system according to the present invention was tested both, in a known standard dissolution test as well as in a test, which has been adapted to the digestive conditions that are representative for ectothermic salmonid fish. Firstly, release of blue dextran from Ca-alginate and EDA-alginate was assessed in vitro in a standard dissolution test (pH 1.2 and pH 6.8 at 37° C.). Secondly, release from the same alginates was tested in alkaline dissolution media of pH 8.0 and pH 8.6 at 18° C. Based on the results from the previous tests, a new dissolution test strategy (pH 3.0 followed by pH 8.6 at 4° C. or at 18° C.) was developed. Finally, release of horse-radish peroxidase (HRP) from both Ca-alginate and EDA-alginate was assessed in vivo in a feeding trial to prove the suitability of ethylenediammonium alginate as an oral delivery system for macromolecules in fish.
[0066] 4.1 Materials used in the experiments
[0067] Sodium alginate (Protanal LF 20/40) was obtained from FMC BioPolymer AS (Norway). Horseradish Peroxidase (HRP, P/N 31491, Thermo Fisher Scientific) was acquired from Perbio Science UK Ltd. Deionised water (DI water, analytical reagent) was purchased from Fishe Chemical Ltd. (UK). The following reagents were purchased from VWR Ltd (UK): calcium chloride dihydrate (AnalaR NORMAPUR®, ACS analytical reagent), sodium bicarbonate (AnalaR NORMAPUR®, ACS analytical reagent), sodium hydroxide (>98%, flake, Alfa Aesar), potassium hydrogen phthalate (>99%, Alfa Aesar) and hydrochloric acid (37%, AnalaR NORMAPUR®, analytical reagent). The following reagents were purchased from Sigma-Aldrich Ltd. (UK): ethylenediamine dihydrochloride (98%, Aldrich), glycine (>99%, ReagentPlus®), disodium phosphate dihydrate (?99%, analytical reagent), monosodium phosphate dihydrate (?98%, analytical reagent), blue dextran, BD (M.W=2,000,000; [reactive blue 2]=0.10 to 0.12 mmol g.sup.−1 dextran) and 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate system (ready to use). EWOS Opal 200 base pellet (BP, 5.8% fat) was produced at Technology Centre of EWOS Innovation AS in Dirdal (Norway). Fish oil (EWOS ID: 20180) was acquired from Egersund Sildolj efabrikk AS (Egersund, Norway).
[0068] Stock solutions:
[0069] Blue dextran encapsulation formulation (BD-EncForm) was prepared by dissolving sodium alginate (2.0% w/v) in a BD solution (50.0 mg m1.sup.−1) at ambient temperature.
[0070] Buffer pH 1.2: Sodium chloride/hydrochloric acid (NaCl/HCl, pH 1.2) buffer was prepared by mixing NaCl solution (250 ml, 23.38 g 1.sup.i, 0.4M NaCl) with 0.4M HCl solution (425 ml). Before making up the volume to 1000 ml with DI water, the pH of the buffer solution was adjusted to 1.2.
[0071] Buffer pH 3.0: Potassium hydrogen phthalate solution (500 ml, 81.69 g 1.sup.1, 0.4M KPH) was combined with HCl solution (223 ml, 0.4M HCl) to produce stock solution of KPH/HCl buffer (pH 3.0). The buffer solution was replenished with DI water to a total volume of 1000 ml.
[0072] Buffer pH 6.8: Monosodium phosphate dihydrate solution (255 ml, 62.40 g 0.4M NaH2PO4.2H2O) was mixed with disodium phosphate dihydrate solution (245 ml, 71.20 g 1.sup.−1, 0.4M Na2HPOa2H2O) to form phosphate buffer (pH 6.8). The resulting buffer was adjusted to pH 6.8 before levelling the volume to 1000 ml with DI water.
[0073] Buffer pH 8.0: Phosphate buffer (pH 8.0) was made by combining monosodium phosphate dihydrate solution (26.5 ml, 62.40 g 1.sup.i, 0.4M NaH2POaH2O) with disodium phosphate dihydrate solution (473.5 ml, 71.20 g 1.sup.i, 0.4M Na2HPOa2H2O). The resulting buffer solution was adjusted to pH 8.0 and then diluted with DI water to a final volume of 1000 ml.
[0074] Buffer pH 8.6: Glycine solution (250 ml, 30.03 g 1.sup.i, 0.4M Gly) was mixed with sodium hydroxide solution (20 ml, 16 g 1.sup.i, 0.4M NaOH) to produce Gly/NaOH buffer. After adjusting the pH to 8.6, the generated buffer solution was diluted to a volume of 1000 ml with DI water.
[0075] Sodium bicarbonate (150 g 1.sup.i, NaHCO.sub.3) was dissolved in DI water to produce a saturated NaHCO.sub.3 solution. Any undissolved NaHCO.sub.3crystals were filtered off prior to use.
[0076] Horseradish Peroxidase (HRP) encapsulation formulation (HRP-EncForm) was prepared by dissolving sodium alginate (2.0% w/w) in an HRP solution (400 pg m1.sup.−1 of DI water) at 4° C.
[0077] HRP stock solution (5 mg m1.sup.−1) was prepared by dissolving HRP powder (10.0 mg) in DI water (2.0 ml). Aliquots of this HRP solution (20.0 μl) were stored at −20° C. prior to constructing the standard curves in the enzyme assay.
[0078] The cross-linking solutions (CaCl.sub.2) sol. and EDA2HC1 sol.) were prepared by separately dissolving calcium chloride dihydrate (36.8 g 1.sup.i, 0.25M CaCl2.2H2O) and ethylenediamine dihydrochloride (33.3 g 1.sup.i, 0.25M EDA2HC1) in DI water.
[0079] 4.2 In vitro test of dissolution of alginate microbeads
[0080] Blue dextran (BD) was selected as a model compound for the simulated release of active pharmaceutical ingredients (API) from alginate matrices.
Preparation of alginate beads loaded with blue dextran
[0081] BD-EncForm ([BD]=50.0 mg m1.sup.−1) was extruded from a 60 ml plastic syringe (BD PlastipakTM) through a needle (i.d. =2.0 mm) into the cross-linking solutions. Flow rate (50.00 m111.sup.−1) was maintained constant by a syringe pump (Harvard PHD4400, Harvard Apparatus Ltd, Edenbridge, UK) working in the volume mode (target volume=1.50 ml). This set up was used to extrude 80 batches of BD-EncForm a 1.50 ml each. The cross-linking solutions, CaCl.sub.2) sol. (40×10.0 ml) and EDA2HC1 sol. (40×10.0 ml) were used to yield BD-Ca-alg (40 batches) and BD-EDA-alg beads (40 batches) respectively. Common to each batch was that alginate beads were separated from the cross-linking solution 10 min after the last bead was generated. Thereafter, the recovered alginate beads loaded with BD were washed with DI water (3×5 ml). BD-Ca-alg (10 batches) and BD-EDA-alg beads (10 batches) were used to determine encapsulation efficiency while the remaining batches were utilised in dissolution tests. The particle size distribution and shape were determined by a stereo microscope (Leica MZ10 F, Leica Microsystems, Wetzlar, Germany).
Encapsulation Efficiency of Blue Dextran
[0082] BD-Ca-alg (10 batches) and BD-EDA-alg (10 batches) beads were added to saturated NaHCO.sub.3 solution (20×9.0 ml, pH=8.0). As a result, all beads were completely dissolved after two hours under stirring. To equalise all sample volumes, the level of the resulting solutions was adjusted to 10 ml with DI water. These newly created solutions (n=20) were filtered through syringe filters (0.45 VWR) prior to application onto a 96-well polystyrene plate (Nunc™, Sigma-Aldrich) for an endpoint assay. Absorbance of BD was measured using a VERSAmax microplate reader (Molecular Devices LLC, Sunnyvale, Calif., USA) at 610 nm and 24° C. BD concentration of the samples was determined by using standard curve in the range of 1.0 to 3.9×10.sup.−3 mg m1.sup.−1. Standard curves were generated by plotting BD concentrations of nine two-fold serial dilutions of BD solution (1.0 mg m1.sup.−1) versus absorbance. The BD solution (1.0 mg m1.sup.−1) was derived from BD-EncForm ([BD]=50.0 mg m1.sup.−1) using saturated NaHCO.sub.3 solution as a diluent.
A. Standard Dissolution Test
[0083] Dissolution tester (Caleva 8ST, Caleva International Ltd, UK) equipped with dissolution vessels (V=1000 ml) and rotating stainless steel baskets (40 mesh) was used to assess BD release from the two different types of alginate beads. This setup was in compliance with the standard requirements for Apparatus 1 set by United States Pharmacopoeia (USP) and described in the General Chapter <711>.
[0084] Dissolution medium (300 ml; NaCl/HCl buffer, pH 1.2 or phosphate buffer, pH 6.8) was added into each of the vessels and then allowed to temperate overnight at 37° C. The dissolution experiment started within 2 h after producing alginate beads according to the method described above. The alginate beads (BD-Ca-alg or BD-EDA-alg) were placed into rotating baskets (100 rpm) and then submerged into dissolution vessels. The following experiments were carried out in quintuplicate at 37° C.: Test 1) BD-Ca-alg at pH 1.2, Test 2) BD-EDA-alg at pH 1.2, Test 3) BD-Ca-alg at pH 6.8 and Test 4) BD-EDA-alg at pH 6.8.
[0085] Samples (1.0 ml sample.sup.−1) were taken from the vessel at five minutes intervals through an extent of 50 minutes. An equivalent volume of dissolution medium (1.0 ml, 37° C.) was then added to keep the liquid level in the vessels constant. All samples were filtered through a syringe filter (0.45 VWR) before placing them onto a 96-well polystyrene plate (Nunc™ Sigma-Aldrich) for an endpoint assay. Reading of the plate was performed by a VERSAmax microplate reader (Molecular Devices LLC, Sunnyvale, Calif., USA) at 610 nm and 24° C. Amount of BD released was determined by applying standard curve approach. Standard curves were generated by plotting BD concentrations of nine two-fold serial dilutions of BD solution (1.0 mg m1.sup.−1) versus absorbance in the range of 1.0 to 3.9×10.sup.−3 mg m1.sup.−1. The BD solution (1.0 mg m1.sup.−1) was derived from BD-EncForm ([BD]=50.0 mg m1.sup.−1) using the experimental buffer as a diluent.
[0086] B. Assessment of alkaline dissolution media
[0087] In order to select an alkaline dissolution medium, which is representative for the salmon intestine in terms of temperature and pH, four dissolution tests were carried out as described in the previous section. The following experiments were performed with five replications each in dissolution media (phosphate buffer, pH 8.0 and Gly/NaOH buffer, pH 8.6) at 18° C.: Test 5) BD-Ca-alg at pH 8.0, Test 6) BD-EDA-alg at pH 8.0, Test 7) BD-Ca-alg at pH 8.6, and Test 8) BD-EDA-alg at pH 8.6. The concentration of BD in the samples was determined in the same way as previously described in the standard dissolution test.
[0088] C. A new dissolution test strategy adapted to fish
[0089] The applied conditions in this test were in accordance with the conditions that alginate beads could typically be exposed to during their passage through gastrointestinal tract of A. salmon. Accordingly, alginate beads were first submerged into acidic dissolution medium (KPH/HCl buffer, pH 3.0) for 15 min and then the acidic buffer solution was replaced with an alkaline dissolution medium (phosphate buffer, pH 8.0). In this test, the same UPS Apparatus 1 as previously described was used. In order to replicate the natural conditions in which A. salmon lives as fully as possible, two different temperatures (4° C. and 18° C.) were applied in the test. The chosen temperatures typically correspond to the water temperatures during summer and winter at the Norwegian coast. For this reason, the following four dissolution tests were carried out in quintuplicate: Test 9) BD-Ca-alg at pH 3.0-8.0 and 4° C., Test 10) BD-EDA-alg at pH 3.0-8.0 and 4° C., Test 11) BD-Ca-alg at pH 3.0-8.0 and 18° C., Test 12) BD-EDA-alg at pH 3.0-8.0 and 18° C. The first sample was taken after 15 min in the KPH/HCl buffer (pH 3.0). Further sampling, which started 5 min after replacing the dissolution medium, was conducted as previously described in the standard method. Similarly, the endpoint assay was carried out according to the method described in in the preceding sections. Encapsulation efficiency of BD was taken in consideration when calculating percentage release from alginate beads.
[0090] Statistical analysis
[0091] Dissolution profiles of BD shown as curves of the mean percentage of cumulative BD release with error bars (95% confidence intervals) over time were generated using Data analysis and Scatter plot functions in Microsoft Excel 2010.
[0092] Results
[0093] The average encapsulation efficiency of BD in BD-Ca-alg and BD-EDA-alg was 90% (SD=4%) and 70% (SD=4%), respectively. Mean size of the Ca-alginate beads was 3.0 mm while the mean size of the EDA-alginate beads was 3.7 mm. The shape of the beads was spherical for both types of beads (
[0094] Release of BD from both Ca-alginate and EDA-alginate beads was below the quantification limit (LoQ=3.9×10.sup.−3 mg m1.sup.−1) of the assay in the standard dissolution tests (Test 1 and 2 performed in pH 1.2 at 37° C.; Test 3 and 4 performed in pH 6.8 at 37° C.).
[0095] Similarly, the release of BD from both alginate beads was below LoQ in the tests carried out in the alkaline dissolution media (Test 5 and Test 6 performed in pH 8.0 at 18° C.; Test 7 and Test 8 performed in pH 8.6 at 18° C.). From the present in vitro study it could be seen that both Ca-alginate and EDA-alginate matrix are poorly soluble in buffers with pH 1.2 at 37° C., pH 6.8 at 37° C., pH 8.0 at 18° C. and pH 8.6 at 18° C. within a time frame of 50 minutes.
[0096] In the dissolution tests (Test 9-12) adapted to the conditions in which salmonid fish digest alginate, significant release of BD was observed in all four experiments. The release, which was below LoQ at pH 3.0, was fairly rapid after raising the pH from pH 3.0 to pH 8.0. From Test 12 at 18° C. (
TABLE-US-00001 TABLE 1 Dissolution curves related to the release of blue dextran release (BD) from calcium (BD-Ca-alg) and ethylenediammonium alginate (BD-EDA-alg) at 4° C. and 18° C. First- and second-order approximations of dissolution curves are shown with corresponding coefficients of determination (R.sup.2). Data fits very well both linear and non-liner function in the time interval 0 to 50 minutes after changing dissolution medium. Product Order Dissolution curves R.sup.2 BD-EDA-alg 1.sup.st y = 2.2101x − 3.9877 0.97 at 18° C. 2.sup.nd y = −0.0112x.sup.2 + 2.7696x − 8.1836 0.98 BD-EDA-alg 1.sup.st y = 2.1061x + 2.251 0.96 at 4° C. 2.sup.nd y = −0.0264x.sup.2 + 3.4249x − 7.6404 0.99 BD-Ca-alg 1.sup.st y = 1.8109x + 1.7095 0.99 at 18° C. 2.sup.nd y = −0.01x.sup.2 + 2.3088x − 2.0247 1.00 BD-Ca-alg 1.sup.st y = 0.5760x + 4.0063 0.95 at 4° C. 2.sup.nd y = −0.0067x.sup.2 + 0.91121x + 1.4852 0.98
[0097] Drugs, which are encapsulated in alginate matrices, are principally released by two mechanisms: 1) diffusion of the drug through the pores of the polymer network and 2) degradation of the polymer network. Unlike low molecular weight drugs, blue dextran (M.W. =2,000,000) is so large that it cannot diffuse through the pores of an alginate matrix without their further expansion (Kim, C. and Lee, E., 1992. The controlled release of blue dextran from alginate beads. Int. J. Pharm. 79, 11-19). Therefore, most of the BD release from alginate matrices presumably happens due to degradation of the polymer network. In earlier studies a very low release rate of fluorescein isothiocyanate dextran (M.W. =145,000) was observed at pH 7.4 during the first hour.
[0098] Very little degradation of EDA-alginate is expected to occur at pH 3.5-7.0 since most of the ethylenediamine (uncharged form) will exist as ethylenediammonium (positively charged) at a pH<7.0 and most of the alginate (alginic acid) will be deprotonated (negatively charged) at pH >3.5.
[0099] According to the experimental results presented in the present invention, an effective way to weaken the cross-linked structure of an alginate matrix is to use an acidic dissolution medium (pH<3.5) before applying an alkaline medium (pH >7.0). Without being bound to theory, the following mechanism may apply: At a pH below 3.5 (alginic acid, pKa=1.5-3.5), hydrogen ions (H.sup.+) apparently replace ethylenediammonium in alginate networks yielding an unlinked structure of alginic acid. Although alginic acid is poorly water soluble, it is easily convertible to an aqueous soluble form (e.g., sodium alginate) at higher pH. At the same time, an increasing alkaline pH (pH >7.0) is reducing the crosslinking strength of ethylenediammonium through deprotonation of its amine groups and conversion to ethylenediamine, the uncharged form. As a consequence, a foregoing treatment with acidic medium (pH<3.5) may give much faster release rate than the direct treatment with an alkaline medium (pH >7.0). Therefore, the standard conditions commonly applied in dissolution tests (pH=1.2 and 6.8 at 37° C.) very often result in very slow dissolution rates.
[0100] By subsequent application of media with pH=3.0 followed by pH=8.0, the high release rates shown in the
[0101] It is generally known that the dissolution of cross-linked alginates is pH-dependent and temperature dependent. From the
4.3 In Vivo Test of Dissolution of Alginate Microbeads
[0102] Performance of ethylenediammonium alginate as an oral delivery system according to the present invention was further tested in an in vivo experiment with Atlantic salmon (Salmo salar).
Preparation of Alginate Microbeads Loaded with HRP:
[0103] Calcium alginate (HRP-Ca-alg) and ethylenediammonium alginate (HRP-EDA-alg) microbeads loaded with HRP were produced in an encapsulation process referred as aerodynamically assisted jetting. In the process of aerodynamically assisted jetting, jet of alginate droplets was generated by extruding HRP-EncForm (2×240.0 g) through a jetting head by means of air pressure. The jetting head was fitted with a nozzle (0=500 μm) and an exit orifice (0=500 μm) of the same size. The created jet was directed towards the surface of a cross-linking solution from a distance of 100 mm. The cross-linking solutions, CaCl.sub.2) sol. (500 ml, 4° C.) and EDA2HC1 sol. (500 ml, 4° C.) were used to yield HRP-Ca-alg (240.0 g) and HRP-EDA-alg (240.0 g) microbeads, respectively. The flow rate (100 ml h.sup.−1) of the alginate solution was controlled by a high precision syringe pump (PHD 4400, Harvard Apparatus Ltd, Edenbridge, UK). The air pressure in the process was maintained at 3.00 bar by a precision regulator (IR1000, SMC Corporation, Tokyo, Japan). The resulting microbeads were filtered off by suction filtration and washed with DI water (3×20 ml) before storing at −20° C. The obtained mass of HRP-Ca-alg and HRP-EDA-alg microbeads was 81.54 g and 78.29 g, respectively. Laser diffraction system (HELOS BR CUVETTE, CUV-50ML/US, optical module R5, Sympatec GmbH, Clausthal-Zellerfeld, Germany) was used to measure the size of generated alginate beads at wavelength λ=632.8 nm. The median diameter of the microbeads was 25-26
[0104] If the obtained beads shall be incorporated into a feed pellet via the surface, than the diameter of the microbeads must correspond to the pore size of the pellet. This is achieved by the above-described method.
Preparation of the Experimental Feeds
[0105] Two experimental feeds (5.0 kg a batch) comprising HRP were produced by applying HRP-alg microbeads (d.sub.median=25-26 μm) suspended in fish oil (1210.0 g) to EWOS Opal 200 base pellet (BP) in a vacuum infusion coating process (Table 2).
TABLE-US-00002 TABLE 2 Composition of the experimental feeds HRP-Ca-feed, HRP-EDA-feed and Ctrl-feed Oil mixture Fish HRP-Ca- HRP-EDA- Feed oil alg.sup.i alg.sup.ii BP.sup.iii Total name [g] [g] [g] [g] [g] Ctrl-feed 1210.00 0.00 0.00 3790.00 5000.00 HRP-Ca-feed 1210.00 81.54 0.00 3708.46 5000.00 HRP-EDA-feed 1210.00 0.00 78.29 3711.71 5000.00 .sup.iHRP-Ca-alg - product generated by encapsulating HRP solution into Ca-alginate matrix .sup.iiHRP-EDA-alg - product made by encapsulating HRP solution into EDA-alginate matrix .sup.iiiBP - base pellet is a semi-finished fish feed product, dry extrudate lacking oil mix
[0106] HRP-Ca-feed was prepared by coating BP (3708.46 g) with oil suspension containing HRP-Ca-alg (81.54 g) while HRP-EDA-feed was made ready by applying oil suspension containing HRP-EDA-alg (78.29 g) to BP (3711.71 g) in a vacuum coater. Control feed (Ctrl-feed) without HRP was produced by coating BP (3790.00 g) with fish oil (1210.00 g) only.
[0107] Fish trial
[0108] Atlantic salmon Salmo salar (Total number: n=495, mav.sub.g=394 g) were distributed randomly among nine circular seawater tanks (d=1 m, V=0.5 m.sup.3, t.sub.water=5° C.) at EWOS Innovation AS (Dirdal, Norway) nine weeks prior to the start of the trial. The tanks were randomly divided into three groups (Ca-alginate, EDA-alginate and Control), with three tanks being assigned to each group. After a nine weeks long acclimatisation stage, the Ca-alginate and EDA-alginate group of fish were treated with HRP-Ca-feed and HRP-EDA-feed, respectively. The Control group was fed Ctrl-feed. The treatment lasted for two weeks after which followed a sampling. The health status of the fish was very good during the 11 weeks long span of the trial.
Sampling
[0109] Prior to sampling, fish (n=15 fish per tank) were anaesthetised with Finquel® (100 mg 1.sup.−1). Weight of individual fish was recorded for each sampled fish. The following samples were collected 1) Stomach, 2) Pyloric Caeca, 3) Mid intestine, and 4) Distal intestine. Each of the sampled gastrointestinal compartments was opened by longitudinal incision and placed into a container with DI water (10.0 ml, t=4° C.). After vigorous shaking, the solid content was separated from the liquid phase by gravity filtration. The resulting filtrate (2 ml) from each container was transferred to an Eppendorf tube and stored at −20° C. until assayed.
[0110] Sample analysis
[0111] The samples were thawed and spun down at 4000 rpm for four minutes prior to use. Stomach and pyloric caeca samples were applied undiluted while mid and distal intestine samples were diluted 1:200 with DI water (4° C.) before assaying. For diluted mid and distal intestine samples (1:200) below quantification limit (0.391 ng m1.sup.−1), lower dilutions like 1:10 and 1:100 or no dilution were used to increase sensitivity. Kinetic assay was carried out by pipetting aliquots of samples (50 μl) onto 96-well polystyrene plates (Nunc™, Sigma-Aldrich). Reaction was initiated by the addition of 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate (50 37° C.). Absorbance measurements were made using a VERSAmax microplate reader (Molecular Devices LLC, Sunnyvale, Calif., USA) at 655 nm. Kinetic rates were recorded every 20 seconds for a total of 10 minutes at 37° C. HRP concentration of the samples was determined by using standard curve as a quantification tool in the range of 0.391 to 200 ng m1.sup.−1. A standard curve was generated by plotting HRP concentrations of 10 two-fold serial dilutions of HRP solution (200 ng m1.sup.−1) against their kinetic rates (slope of the absorbance versus time curves).
[0112] Statistical analysis
[0113] Mean HRP concentrations with error bars (95% confidence intervals) found in different compartments of gastrointestinal tract were calculated and represented in a graph using IBM SPSS Statistics for Windows, version 22.0.
[0114] Results
[0115] In the current trial, a fish consumed 12-13 g feed on average during the treatment with feed containing alginate encapsulated HRP. As a result, the treated fish (mav.sub.g=490−500 g) received between 230 and 240 μg of HRP in the period (Table 3).
TABLE-US-00003 TABLE 3 HRP dose related to the fish size in unit of mass and the weekly feed intake (FI) per fish. The dose is shown as a weekly HRP dose per fish (μg fish.sup.−1 week.sup.−1) and a weekly HRP dose per unit of fish mass (μg fish.sup.−1 week.sup.−1). Feeds used: without HRP (Ctrl-feed), with calcium alginate encapsulated HRP (HRP-Ca-feed), ethylenediammonium alginate encapsulated HRP (HRP-EDA-feed). Fish FI HRP dose.sup.i HRP dose size (g fish.sup.−1 (μg fish.sup.−1 (μg kg.sup.−1 Group Feed (g) ± SD week.sup.−1) ± SD week.sup.−1) ± SD week.sup.−1) ± SD Control Ctrl-feed 492 ± 121 12.7 ± 1.7 0.00 0.00 Ca-alginate HRP-Ca-feed 495 ± 95 12.5 ± 1.4 239 ± 26 483 ± 46 EDA-alginate HRP-EDA-feed 499 ± 108 12.1 ± 0.9 232 ± 17 465 ± 50 .sup.iHRP dose is theoretical and assumes very little loss due to processing.
[0116] The group of fish (Ca-alginate) fed HRP-Ca-feed showed significantly higher concentration of HRP in the distal intestine compared to the other gastrointestinal (GI) compartments (
[0117] From the
[0118] In conclusion, by way of the present invention it was not only shown that differences do exist between differently cross-linked alginate matrices, but also a novel, more efficient oral delivery systems could be identified. Differences between the EDA-alginate and Ca-alginate are particular evident with respect to dissolution rates at low temperatures. This is highly relevant for ectothermic organisms living at low temperatures such as the Atlantic salmon. In order to recognize these characteristics of alginate beads, the above mentioned new dissolution test strategy has been developed. This new strategy makes the dissolution test highly representative for gastrointestinal conditions found in fish which have a stomach. As a consequence, the results generated by the redesigned dissolution test are in strong correlation with the results obtained from the present in vivo study. On the whole, EDA-alginate is an excellent delivery system for macromolecular drugs to ectothermic animals like salmon. Furthermore, there is convincing evidence that this delivery system is surprisingly temperature-independent within the temperature range of salmon's life habitat. The practical implication of the results of the present study is that the amount of drug delivered is irrespective of the environmental temperature when using this novel oral delivery system.
[0119] The advantage of ethylenediammonium alginate micro beads as oral delivery system when incorporated in fish feed and orally administered to fish, is that the beads efficiently dissolve in the fish intestine and deliver the content at the right place for absorbance in the digestive conditions as typically found in fish. However, the present invention, even though being found particularly suitable for use in fish, is not restricted to this group of organisms and may likewise also be used as oral delivery systems for macromolecules in mammals including humans, amphibians, reptiles, birds, crustaceans, molluscs, etc.
[0120] Bioactive agent in accordance with the present invention includes any drug, substance, compound, composition or mixture thereof, which are effective in therapy or prophylactic treatment in organisms and which are suitable for encapsulation in alginate and following oral delivery. This includes agents such as proteins, peptides, vaccines, antibodies, antigens, hormones, drugs, particularly macromolecular drugs, amino acids, nucleotides, polynucleotides, enzymes, any physiologically active substance, nutrients, prebiotics, probiotics, immune stimulants and the like.
[0121] In the context of the present invention, by the term “cold water” organism is meant any ectothermic animal which typically lives in average environmental temperature of about 20° C. or lower. Likewise a “cold water fish” is a fish living in average water temperatures of about 20° C. or lower. Typical examples for cold water fish are cold water marine fish such as codfish and salmonids.
[0122] By the term “short intestinal tract” or “short intestine” in relation to a fish is meant that the total length of the intestine is no more than about 2.5 times the body length of said fish.
[0123] It will be appreciated that the features of the invention described in the foregoing can be modified without departing from the scope of the invention as defined in the accompanying claims.