A PHARMACEUTICAL COMPOSITION IN ORAL DOSAGE FORM

Abstract

A pharmaceutical composition in an oral dosage form for use in a method of treating diabetes in a subject is described. The oral dosage form comprises gastric-resistant ileal-sensitive microcapsules comprising a matrix insulin contained within the matrix, in which the matrix comprises denatured whey protein, and in which the oral dosage form is administered orally to the subject. The microcapsules may be cold-gelated and vacuum dried microcapsules, and thus are subject to less heat treatment than convention drug-containing microparticles. The microcapsules may be provided as agglomerates of microcapsules, generally having an average particle size of 1-2 microns. Agglomerates of microcapsules are suitable for slow release of active agent in the ileum.

Claims

1. A pharmaceutical composition in an oral dosage form comprising gastric-resistant ileal-sensitive microcapsules comprising a matrix and insulin contained within the matrix, in which the matrix comprises denatured whey protein, in which the microcapsules are cold-gelated and vacuum dried microcapsules.

2. A pharmaceutical composition of claim 1, in a unit dose form comprising 10-100 IU of insulin.

3. A pharmaceutical composition of claim 1 or 2, in which the microcapsules comprise as a dry weight %: 90 to 99% denatured whey protein; and 1 to 10% insulin.

4. A pharmaceutical composition of any of claims 1 to 3, in which the microcapsules have an average particle size of 200 to 500 microns.

5. A pharmaceutical composition of any of claims 1 to 4, in which the matrix of the microcapsules is free of polysaccharide gelling agent.

6. A pharmaceutical composition of any of claims 1 to 5, that is free of chitosan, surfactant-permeation enhancer, and cell penetrating peptides.

7. A pharmaceutical composition of any of claims 1 to 6, in which the matrix consists essentially of denatured whey protein.

8. A pharmaceutical composition of any of claims 1 to 7, in which the microcapsules have a water activity (Aw) of less than 0.25.

9. A pharmaceutical composition of any of claims 1 to 7, in which the microcapsules have a water activity (Aw) of 0.20 or less.

10. A pharmaceutical composition of any of claims 1 to 7, in which the microcapsules have a water activity (Aw) of 0.15 or less.

11. A pharmaceutical composition of any of claims 1 to 10, in which the oral dosage form is a powder.

12. A pharmaceutical composition of any of claims 1 to 10, in which the oral dosage form is a tablet formed by compression of the microcapsules.

13. A pharmaceutical composition of any of claims 1 to 10, in which the oral dosage form is a tablet formed by direct compression of the microcapsules.

14. A pharmaceutical composition of any of claims 1 to 13, for use in a method of treating diabetes in a subject, in which the oral dosage form is administered orally to the subject.

15. A pharmaceutical composition of any of claims 1 to 13, for use of claim 14, in which the blood glucose levels of the subject are reduced by at least 15% within 4 hours of administration of the pharmaceutical composition to the subject.

16. A pharmaceutical composition of any of claims 1 to 13, for use of claim 14, in which the blood glucose levels of the subject are reduced by at least 25% within 4 hours of administration of the oral dosage form to the subject.

17. A method of making microcapsules comprising the steps of providing microdroplets comprising denatured whey protein and insulin by extrusion; curing the microdroplets by immersion in a curing bath to form microcapsules having a matrix comprising denatured whey protein and insulin contained within the matrix; removing the microcapsules from the curing bath; and vacuum drying the microcapsules.

18. A method according to claim 17, in which the microdroplets are generated using concentric nozzles by extruding a suspension or solution of the insulin and denatured whey protein through an inner nozzle and simultaneously extruding a denatured whey protein suspension through an outer nozzle.

19. A method according to claim 17, in which the microdroplets are generated by extruding a suspension comprising denatured whey protein and insulin through a single nozzle.

20. A method according to any of claims 17 to 20, in which the suspension comprising denatured whey protein comprises 10-20% denatured whey protein.

21. A method according to any of claims 17 to 20, in which the step of providing the microdroplets comprises the steps of: dissolving insulin in an acid to provide an insulin solution; combining the insulin solution with a buffer to provide a buffered insulin solution with a pH to 6-8; and combining the buffered insulin solution with the suspension comprising denatured whey protein prior to the extrusion step.

22. A method according to claim 21, in which the buffered insulin solution is added to the suspension comprising denatured whey protein dropwise.

23. A method according to any of claims 17 to 22, in which the microcapsules are subjected to a first vacuum drying step and a second vacuum drying step.

24. A method according to any of claims 17 to 23, in which the microdroplets consist essentially of denatured whey protein, insulin and solvent.

25. A method according to any of claims 17 to 24, in which the microcapsules prior to drying have a water activity (Aw) of 0.993-0.997.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0099] FIG. 1A shows microscope image of vacuum dried insulin loaded microcapsules according to the invention.

[0100] FIG. 1B shows (human eye visual) homogenous size distribution of a batch of vacuum dried agglomerates of insulin loaded microcapsules according to the invention.

[0101] FIG. 2A shows (microscope image ×40) even size distribution of vacuum dried agglomerates of insulin loaded microcapsules according to the invention.

[0102] FIG. 2B shows (microscope image ×40) vacuum dried agglomerates of insulin loaded microcapsules according to the invention

[0103] FIG. 2C shows (Scanning Electron Microscope image 10 um) of vacuum dried agglomerates of insulin loaded microcapsules according to the invention

[0104] FIG. 3A shows (Scanning Electron Microscope image 10 um) of vacuum dried agglomerates of insulin loaded microcapsules according to the invention

[0105] FIG. 3B shows (Confocal Scanning Microscope image) of vacuum dried insulin loaded microcapsules (before agglomeration) according to the invention

[0106] FIG. 4 shows agglomerates of fluidised-dried insulin loaded microcapsules according to the invention.

[0107] FIG. 5 shows agglomerates of vacuum-dried insulin loaded microcapsules according to the invention.

[0108] FIG. 6 shows agglomerates of vacuum-dried insulin loaded microcapsules according to the invention.

[0109] FIG. 7 shows the effects of oral dosage of micro-encapsulated insulin (17.7 to 50 IU FIASP) on blood glucose levels (mmol/l) in a Type I diabetes patient. FIG. 7 represents “Units of reduction after 3 h of dosage (expressed in mmol glucose/L; red bold line—) and “% Reduction” (white columns) compared to subcutaneous injection of 5 IU of insulin (control; black, initial column). Ingested insulin microbeads (white columns) were consumed at different doses including 17.9, 25 and 50 units).

[0110] FIG. 8 shows the effects of oral dosage of micro-encapsulated insulin (50 IU FIASP) on blood glucose levels (mmol/l) in a Type I diabetes patient. Figures illustrates “Units of reduction after 3 h of dosage (mmol glucose/L; red line—) and “% Reduction” (black columns) after ingestion of 50 units of micro-encapsulated insulin

[0111] FIGS. 9A and 9B. Fourier-Transform IR spectrophotometric graph (Vector normalised at 1,580-1,710 cm.sup.−1) for denatured protein and comparison with milk protein with various levels of denaturation.

[0112] FIG. 10. Difference in the area under the curve of glucose concentration after ingestion of micro-encapsulated insulin (50 units).

[0113] FIG. 11. Difference in the area under the curve of glucose concentration during and after injected of aspart insulin.

[0114] FIG. 12. HPLC chromatogram to exemplify protein denaturation optimal encapsulation

DETAILED DESCRIPTION OF THE INVENTION

[0115] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0116] Definitions and General Preferences

[0117] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0118] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term “a” or “an” used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.

[0119] As used herein, the term “comprise,” or variations thereof such as “comprises” or “comprising,” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term “comprising” is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

[0120] As used herein, the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, age, poisoning or nutritional deficiencies.

[0121] As used herein, the term “treatment” or “treating” refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term “therapy”.

[0122] Additionally, the terms “treatment” or “treating” refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term “prophylaxis”.

[0123] As used herein, an effective amount or a therapeutically effective amount of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate “effective” amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure. Improvement may be observed in biological/molecular markers, clinical or observational improvements. In a preferred embodiment, the methods of the invention are applicable to humans, large racing animals (horses, camels, dogs), and domestic companion animals (cats and dogs).

[0124] In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include “individual”, “animal”, “patient” or “mammal” where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human. As used herein, the term “equine” refers to mammals of the family Equidae, which includes horses, donkeys, asses, kiang and zebra.

[0125] As used herein, the term “oral dose form” refers to a pharmaceutical composition formulated for oral administration. Examples include tablets, pills, capsules, thin films, pastes, gels, powders, granules, liquid solutions or suspension. Tablets may be formed by direct compression. The oral dosage form generally includes an active agent and a pharmaceutical excipient. In one aspect of the present invention, the active agent is provided in the form of a microcapsule, in which the active agent is contained within a matrix configured to protect the active agent during gastric transit and release the active agent in the ileum. To this end, the matrix may comprise gelated denatured protein such as whey protein. In one embodiment the oral dosage form is provided as a unit dose, containing a single dose of active agent. In the case of diabetes for example, this may be 10-100 IU of insulin.

[0126] As used herein the term “poorly permeable active pharmaceutical ingredient” refers to an active biopharmaceutical ingredient that is classed as a Class 3 or 4 under the Biopharmaceutical Classification System (BCS) published by the FDA December 2017. Examples include peptide therapeutics such as insulin, desmopressin, octreotide, cyclosporin, vanomycin, salmon calcitonin, semaglutide, exenatide and insulin or an insulin analogue. Insulin may be insulin degludec or insulin aspart.

[0127] The term “microcapsule” as used herein should be understood to mean a particle comprising an active component encapsulated within a matrix comprising denatured whey protein. Preferably, the microcapsule has an average diameter (average particle size) of 200 to 1000 microns. Size is determined using a method of laser diffractometery (Mastersizer 2000, Stable Micro Systems, Surrey, UK). This method is determines the diameter, mean size distribution and D (v, 0.9) (size at which the cumulative volume reaches 90% of the total volume), of micro-encapsulates with diameters in the range of 0.2-2000 μm. For insulin microcapsule size analysis, micro-encapsulate batches were re-suspended in Milli-Q water and size distribution is calculated based on the light intensity distribution data of scattered light. Measurement of microencapsulate size is performed at 25° C. and six runs are performed for each replicate batch (Doherty et al., 2011) (Development and characterisation of whey protein micro-beads as potential matrices for probiotic protection, S. B. Doherty, V. L. Gee, R. P. Ross, C. Stanton, G. F. Fitzgerald, A. Brodkorb, Food Hydrocolloids Volume 25, Issue 6, August 2011, Pages 1604-1617). A preferred method of producing the microcapsules in by extrusion through a nozzle, typically a vibrating nozzle, and curing (gelation) in a gelation bath. In one embodiment, the suspension is sprayed through a nozzle and laminar break-up of the sprayed jet is induced by applying a sinusoidal frequency with defined amplitude to the spray nozzle. Examples of vibrating nozzle machines are and EnCapsulator (Inotech, Switzerland), or equivalent scale-up version such as those produced by Brace GmbH or Capsulae and the like. Typically, the spray nozzle has an aperture of between 100 and 600 microns, preferably between 150 and 400 microns, suitably about 300 microns. Suitably, the frequency of operation of the vibrating nozzle is from 900 to 4000 Hz. Generally, the electrostatic potential between nozzle and acidification bath is 0.85 to 1.8 V. Suitably, the amplitude is from 4.7 kV to 7 kV. Typically, the falling distance (from the nozzle to the curing bath) is less than 100 cm, preferably less than 80 cm, suitably between 50 and 70 cm, preferably between and 65 cm, and ideally about 55 cm. The flow rate of suspension (passing through the nozzle) is typically from 3.0 to 120 ml/min; an ideal flow rate is dependent upon the nozzle size utilized within the process.

[0128] “Cold-gelated” as applied to microcapsules refers to microcapsules having a gelated protein matrix formed by extrusion through a nozzle and curing (gelation) is a gelation bath. Cold gelated microcapsules may be extruded through a single nozzle (in which the matrix contains pockets of active agent) or through a double concentric nozzle which form core-shell type microcapsules. Method of producing microcapsules by cold-gelation is described in WO2010/119041, WO2014/198787 and WO2016/096929.

[0129] “Gastro-resistant”: means that the microencapsulates can survive intact for at least 60 minutes in the simulated stomach digestion model described in Minekus et al., 1999 and 2014 (A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation product, Minekus, M., Smeets-Peeters M, Bernalier A, Marol-Bonnin S, Havenaar R, Marteau P, Alric M, Fonty G, Huis in′t Veld J H, Applied Microbiology Biotechnology. 1999 December; 53 (1):108-14) and (Minekus et al., 2014, A standardised static in vitro digestion method suitable for food—an international consensus, Minekus, A. et al., Food Function, 2014, 5, 1113).

[0130] “Ileal-sensitive”: means that the microencapsulates are capable of releasing their contents in vivo in the ileum of a mammal.

[0131] “Encapsulation efficiency” means the amount of insulin loaded into the microcapsule carrier. The Encapsulation efficiency is calculated as follows by determining the free insulin concentration, and the total amount of insulin (Initial insulin concentration).

EE %=100−[(Free insulin conc./Initial insuling conc.)×100]

[0132] “Vacuum drying” is the mass transfer operation in which the moisture present in a substance, usually a wet solid, is removed by means of creating a vacuum. In chemical processing industries like food processing, pharmacology, agriculture, and textiles, drying is an essential unit operation to remove moisture. Vacuum drying is generally used for the drying of substances which are hygroscopic and heat sensitive, and is based on the principle of creating a vacuum to decrease the chamber pressure below the vapor pressure of the water, causing it to boil. With the help of vacuum pumps, the pressure is reduced around the substance to be dried. This decreases the boiling point of water inside that product and thereby increases the rate of evaporation significantly. The result is a significantly increased drying rate of the product. The pressure maintained in vacuum drying is generally 0.03-0.06 atm and the boiling point of water is 25-30° C. The vacuum drying process is a batch operation performed at reduced pressures and lower relative humidity compared to ambient pressure, enabling faster drying. “Water activity” (aw) is the partial vapor pressure of water in a substance divided by the standard state partial vapor pressure of water. It is measured by the method described in Carter, B. P., Galloway, M. T., Campbell, G. S., & Carter, A. H. (2015). The critical water activity from dynamic dewpoint isotherms as an indicator of pre-mix powder stability. Journal of Food Measurement and Characterization, 9(4), 479-486. The operator's manual of the equipment used is provided at http://manuals.decagon.com/Manuals/13893_AquaLab %20Pre_Web.pdf Values for water activity (Aw) provided herein are obtained at 25° C. unless stated otherwise.

[0133] “Fluidised air drying” refers to an air-drying approach to control the gentle and homogenous drying of wet solids such as micro-capsules. The intensive mass exchange of air fluidized between micro-capsules makes this method effective and suitable for post-drying of micro-capsules and agglomerated micro-capsules.

[0134] The present invention also provides pharmaceutical compositions. Such compositions comprise an effective amount of microcapsules or agglomerates according to the invention, and a pharmaceutically acceptable excipient or carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans. The term “excipient” refers to a diluent, adjuvant, excipient, or vehicle with which the microcapsules or agglomerates of the invention are administered. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol and water.

EXEMPLIFICATION

[0135] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

Example 1—Microcapsules

[0136] Insulin Buffer Preparation.

[0137] Weight 1 g of insulin into a weight boat.

[0138] Note: the amount of insulin and reagents will depend on the size of the batch needed and

[0139] the availability of reagents.

[0140] Pipette 7.5 mL HCl (0.2M).

[0141] Add insulin to the HCl

[0142] Vortex

[0143] Add insulin dissolved in HCL 0.2 M dropwise to the Phosphate buffer and adjust to pH 7.1-7.3

[0144] Premix Preparation

[0145] Prepare 1:19 parts insulin to WPI slurry and disperse using an Ultraturrax or stomacher. Allow the slurry to sit at 30DegC for 25 minutes. Repeat the Ultraturrax step. Sonicate for 60 seconds. Agitate the slurry at 450 rom at 30DegC for 15 minutes.

[0146] Microbead Generation

[0147] Use the 150 um or 300 um nozzle.

[0148] Use Citrate buffer 0.8 M pH 4.4 (addition of Tween 20-0.002%) and heat to 35DegC.

[0149] Ratio of premix to citrate buffer is specified to generated micro-beads of a specific size (150-450 um)

[0150] Apply a mild agitation at 150 rpm

[0151] Maintain Premix under slight stirring during the process (100 rpm).

[0152] Adjust the pressure to generate a suitable bead chain.

[0153] Frequency and electrode will depend on the viscosity of the premix, time after denaturation, salts utiliised

[0154] Frequency: 900-1500 Hz

[0155] Electrode 2000-40000 V

[0156] Control the process and clean the nozzle/tubing if blocking occurs.

[0157] Once the beads are made, leaving sitting in the citrate buffer for 15 min.

[0158] Washing of Wet Beads

[0159] Sieve and wash beads through 500 um sieve.

[0160] Wash beads in de-ionised water

[0161] Drying—Vacuum

[0162] Transfer beads to vacuum air chamber.

[0163] Dry for 30 Min-12 Hour, depending on the bead size and adjust temperature and vacuum pressure accordingly.

[0164] Agglomerates of microcapsules formed are shown in FIGS. 2 and 3. They have an average particle size of 300-500 microns.

[0165] Drying Non-Vacuum

[0166] Transfer beads to fluidisd air chamber.

[0167] Dry for 1 h-12 Hours, depending on the bead size and adjust flow rate from 0.3-25 units per hour.

[0168] Agglomerates of microcapsules formed are shown in FIGS. 4, 5 and 6. They have an average particle size of 1-1.2 mm.

[0169] Sieving and Micro-Capsule Charactersation

[0170] Once the drying is finalized, sieve the Micro-capsule obtained

[0171] Separate Micro-capsules in the different particle size obtained: >500 um, 300-500 um, <300 um. FIG. 1 illustrates dried Micro-capsules.

[0172] Characterize the required particle size fraction: determine moisture content, quantify insulin using ELISA. Keep the Micro-capsules in a sealed foil bag under dry conditions

Example 2—In-Vivo Study

[0173] Microcapsules formed according to Example 1 were administered to a Type I diabetic male human.

[0174] An in vivo I (acute) study was conducted to evaluate the effect of micro-encapsulated insulin on glucose tolerance and sensitivity in a healthy diabetic (Type 1) and to determine if micro-encapsulated insulin increases physiological uptake of glucose in skeletal muscle of diabetic. Objectives include: [0175] Objective 1—Identify the concentration of peripherally administered micro-encapsulated insulin that increases glucose clearance following an oral glucose load in Type 1 diabetic human [0176] Objective 2—Identify whether changes in skeletal muscle glucose uptake are implicated buy micro-encapsulated insulin induced enhancement of glucose clearance in in Type 1 diabetic human.

[0177] Microcapsules formed according to Example 1 were administered to a Type I diabetic male human.

[0178] The microcapsules were administered at a dosage of from 17.9 IU aspart insulin (0.1 g agglomerated microcapsules) to 50 IU aspart insulin (0.2 g of agglomerated microcapsules). The composition was administered mid-morning, around 3-4 hours after the morning sub-cutaneous injection when the subject's glucose levels were stable.

[0179] The patient's blood glucose was measured immediately prior to administration and 3 hours after administration. The subject's blood glucose levels were continuously measured with Free Style Libre Senor System. The intervention was finished between 3-4 hours after the intake of the micro-capsules.

[0180] A subcutaneous injection of 5 IU aspart insulin (FIASP—Novo Nordisk) was employed as a positive control.

[0181] Blank microcapsules containing no insulin was used as a negative control.

[0182] FIGS. 7 and 8 and 9 illustrate that the composition of the invention reduced blood glucose levels in the patient by 25-42% within 3 hours of administration. This compares favourably with the positive control that reduced blood glucose in the same patient by 55% over the same time period. The negative control did not result in a reduction in blood glucose levels over the 3-hour period. Difference in the area under the curve of glucose concentration during injected vs micro-encapsulated insulin were recorded as shown in FIG. 10.

Example 3—In-Vivo Study

[0183] Microcapsules formed according to Example 1 were administered to a Type I diabetic male human.

[0184] An in vivo acute study was conducted to evaluate the effect of micro-encapsulated insulin on glucose tolerance and sensitivity in a healthy diabetic (Type 1) and to determine if micro-encapsulated insulin increases physiological uptake of glucose in skeletal muscle of diabetic. Objectives include: [0185] Objective 1—Identify the concentration of peripherally administered micro-encapsulated insulin that increases glucose clearance following an oral glucose load in Type 1 diabetic human [0186] Objective 2—Identify whether changes in skeletal muscle glucose uptake are implicated buy micro-encapsulated insulin induced enhancement of glucose clearance in in Type 1 diabetic human.

[0187] Microcapsules formed according to Example 1 were administered to a Type I diabetic male human. The microcapsules were administered at a dosage of from 50 IU Human recombinant Insulin (0.05 g agglomerated microcapsules) to 100 IU Human recombinant insulin (0.1 g of agglomerated microcapsules). The composition was administered mid-morning when the subjects glucose levels were stable.

[0188] The patient's blood glucose was measured immediately prior to administration and 3 hours after administration using Free Style Libre Senor System.

[0189] A subcutaneous injection of 5 IU aspart insulin (FIASP— Novo Nordisk) was employed as a positive control.

[0190] Blank microcapsules containing no insulin was used as a negative control.

[0191] The composition of the invention reduced blood glucose levels in the patient by 20-50% within 4 hours of administration. This compares favourably with the positive control that reduced blood glucose in the same patient over the same time period.

[0192] The negative control did not result in a reduction in blood glucose levels over the 3-hour period. Difference in the area under the curve of glucose concentration during injected vs micro-encapsulated insulin were recorded as shown in FIG. 11.

Example 4—Insulin Microcapsules, Single Stage Drying

[0193] Hydrate Whey Protein powder (10.5%-11% protein content) in water [0194] Hydrate and measure pH [0195] Denature protein at 92 DegC for 15 seconds [0196] Cool to room temperature to 22 DegC for 18 hours [0197] Measure level of protein agglomeration by HPLC [0198] Hydrate insulin in 0.1N HCl at Room Temperature for 30 minutes to hydrate [0199] Once hydrated, add 1N NaoH to bring to pH 7.2 and bring volume to 50 ml using phosphate saline solution [0200] Hydrate for 45 minutes at Room Temperature [0201] Add insulin dropwise to denatured protein (1:5 or 1:20 or 1:50) [0202] Extrude solution through a single or double nozzle [0203] If double nozzle is used, out nozzle is pure denature protein (10% solids) [0204] Polymerise microcapsules in sodium citrate buffer Ph 4.4-4.7. at 30DegC [0205] Allow solution to polymerise for 2.5 hours at RT in citrate buffer [0206] Wash insulin micro-capsules in water [0207] Dry the material under vacuum at room temperature for 18-24 hours [0208] Remove material [0209] Measure moisture (<7-8% and Aw content (<0.25) [0210] Quantify insulin on HPLC as per usual method [0211] Store at refrigerated temperature

Example 5—Insulin Microcapsules, Double Stage Drying

[0212] Hydrate Whey Protein powder (10.5%-11% protein content) in water [0213] Hydrate and measure pH [0214] Denature protein at 92 DegC for 15 seconds [0215] 10%-12.5% protein content) [0216] Cool to room temperature to 22 DegC for 18 hours [0217] Measure level of protein agglomeration by HPLC [0218] Hydrate insulin in 0.1N HCl at RT for 30 minutes to hydrate [0219] Once hydrated, add 1N NaoH to bring to pH 7.2 and bring volume to 50 ml using phosphate saline solution [0220] Hydrate for 45 minutes at Room Temperature [0221] Add insulin dropwise to denatured protein (1:5 or 1:20 or 1:50) [0222] Solids content from 11%-18% in solution [0223] Extrude solution through a single or double nozzle

[0224] Polymerise microcapsules in sodium citrate buffer Ph4.4-4.7. at 30DegC [0225] Allow solution to polymerise for 2.5 hours at Room Temperature in citrate buffer [0226] Wash insulin micro-capsules in water [0227] Dry the material under vacuum at room temperature for 18-24 hours at <15 mBar with agitation [0228] Remove material and measure moisture (<7-8% and Aw content (<0.25) [0229] Transfer to secondary chamber for secondary drying for further 24-36 hour drying at <10 mBar (2nd stage drying) with/without agitation [0230] Remove material and pack [0231] Measure moisture (<5% and Aw content (<0.2) of finsihed product [0232] Quantify insulin on HPLC as per usual method [0233] Store at refrigerated temperature

EQUIVALENTS

[0234] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.