A COMPOSITON COMPRISING MICROCAPSULES

Abstract

A composition comprising microcapsules in an oral dosage form is described. The oral dosage form comprises gastric-resistant ileal-sensitive microcapsules comprising a matrix and active agent such as probiotic bacterium, contained within the matrix, in which the matrix comprises denatured whey protein, and in which the microcapsules are be coldgelated and vacuum dried microcapsules, and thus are subject to less heat treatment than conventional probiotic-containing microparticles. The microcapsules may be formed by extrusion through a single or double nozzle and are vacuum dried to a water activity (Aw) of 0.30 or less. The microcapsules may be subjected to two separate vacuum drying steps to further reduce the water activity and provide microcapsules with greater stability against moisture, humidity and thermal processes such as pasteurisation and Ultra High Temperatures (i.e. UHT)

Claims

1. A composition comprising gastric-resistant ileal-sensitive microcapsules comprising a matrix and active agent 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 composition according to claim 1 having a water activity (Aw) of less than 0.30.

3. A composition according to claim 1 having a water activity (Aw) of less than 0.25.

4. A composition according to any preceding Claim, in which the active agent comprises probiotic bacteria.

5. A composition claims 4, in which the microcapsules comprise as a % dry weight: 80 to 90% denatured whey protein; and 10 to 20% probiotic bacteria.

6. A composition of any of claims 1 to 5, in which the microcapsules have an average particle size of 200 to 500 microns.

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

8. A method of claim 7, in which the active agent comprises probiotic bacteria.

9. A method of claim 7 or 8, in which the microcapsules are vacuum dried to a water activity of 0.20 or less.

10. A method of claim 7 or 8, in which the microcapsules are vacuum dried to a water activity of 0.15 or less.

11. A method of any of claims 7 to 10, in which the microcapsules are subjected to a first vacuum drying step and a second vacuum drying step.

12. A method according to claim 11, in which the microcapsules are agitated during the first vacuum drying step and optionally not agitated during the second drying step.

13. A method according to claim 11 or 12, in which the first vacuum drying step is performed at a pressure of 10-15 mBar.

14. A method according to claim 13, in which the second vacuum drying step is performed at a pressure of 5-10 mBar.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0070] 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.

[0071] Definitions and general preferences

[0072] 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:

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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”.

[0077] 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”.

[0078] 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).

[0079] 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.

[0080] As used herein, the term “oral dose form” refers to a 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.

[0081] 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 .Math.m. For insulin microcapsule size analysis, micro-encapsulate batches were resuspended 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 45 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.

[0082] “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.

[0083] “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 JH, Applied Microbiology Biotechnology. 1999 Dec;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).

[0084] “Ileal-sensitive”: means that the microencapsulates are capable of releasing their contents in vivo in the ileum of a mammal.

[0085] “Encapsulation efficiency” means the amount of active agent 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).

[00001]$\text{EE \%}\text{=}\text{100}\text{-}\left[\left(\text{Free insulin conc}\text{.}/\text{Initial insuling conc}\text{.}\right)\times 100\right]$

“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 theprinciple 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.

[0086] 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

[0087] 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.

Probioitic Microcpaules - Single Stage Drying

[0088] Hydrate Whey Protein powder (9.0% - 11.0% protein content) in water Hydrate and measure pH [0089] Denature protein at 95.5 DegC for 80-85 seconds Cool to room temperature to 22 DegC for 16 - 20 hours Measure level of protein agglomeration by HPLC [0090] Hydrate probiotics in milk powder or designated media for specified strain powder or designated media for specified strain [0091] Add probiotic to denatured protein (1:5 or 1:9 or 1:20) [0092] Extrude solution through a single or double nozzle If double nozzle is used, outer nozzle is pure denature protein (9% solids) [0093] Polymerise microcapsules in sodium citrate buffer Ph 4.6 - 4.8 at 20 - 30 DegC [0094] Allow solution to polymerise for max 4 hours at RT in citrate buffer [0095] Wash micro-capsules in water and in skim milk powder solution (5-20% solids content [0096] Dry the material under vacuum at room temperature for 18 20 hours Measure moisture (<10% and Aw content (<0.3) Quantify probiotic content as per usual method [0097] Store at room temperature or refrigerated temperature

Probiotic Micropcaules - Double Stage Drying

[0098] Hydrate Whey Protein powder (10.0% - 11.0% protein content) in water Hydrate and measure pH [0099] Denature protein at 95.5 DegC for 80-85 seconds Cool to room temperature to 22 DegC for 16 -20 hours Measure level of protein agglomeration by HPLC [0100] Hydrate probiotics in milk powder or designated media for specified strain [0101] Add probiotic to denatured protein (1:5 or 1:9 or 1:20) [0102] Extrude solution through a single or double nozzle If double nozzle is used, outer nozzle is pure denature protein (9% solids) [0103] Polymerise microcapsules in todium citrate buffer Ph 4.5 - 4.8 at 20 - 30 DegC [0104] Allow solution to polymerise for max 4 hours at RT in citrate buffer [0105] Wash micro-capsules in water and in skim milk powder solution (5-20% solids content [0106] Dry the material under vacuum at room temperature for 18 20 hours [0107] Transfer material to secondary vacuum chamber for secondary drying [0108] Dry for further 12-38 hour drying at <10 mBar (2nd stage drying) with / without agitation Measure moisture (<5% and Aw content (<0.2) Quantify probiotic content as per usual method [0109] Store at room temperature or refrigerated temperature

EQUIVALENTS

[0110] 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.