U.S. patent application number 10/922917 was filed with the patent office on 2005-01-27 for method of preparing biological materials and preparations produced using same.
This patent application is currently assigned to GAINFUL PLAN LIMITED. Invention is credited to Au Yeung, Terence P.Y., Ko, Thomas S.Y..
Application Number | 20050019417 10/922917 |
Document ID | / |
Family ID | 3826756 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050019417 |
Kind Code |
A1 |
Ko, Thomas S.Y. ; et
al. |
January 27, 2005 |
Method of preparing biological materials and preparations produced
using same
Abstract
Methods for preparing products containing moisture-sensitive
materials, including biological materials such as proteins,
peptides or live cells, comprising at least the steps: (i)
providing a coating liquid comprising at least one active, a sugar
polymer and a water soluble/miscible solvent; (ii) providing a
quantity of microparticles comprising at least water soluble gel
forming solid particles; (iii) fluidizing said quantity of
microparticles within a processing chamber of a of a suitable
apparatus to form a fluidized bed of said microparticles; (iv)
spraying said coating liquid onto said fluidized bed from beneath
the fluidized bed to coat said microparticles therewith under
saturated moisture conditions; and (vi) allowing coated
microparticles to dry, are described. Also described are
compositions and uses.
Inventors: |
Ko, Thomas S.Y.; (Montrose,
AU) ; Au Yeung, Terence P.Y.; (Montrose, AU) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
GAINFUL PLAN LIMITED
|
Family ID: |
3826756 |
Appl. No.: |
10/922917 |
Filed: |
August 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10922917 |
Aug 23, 2004 |
|
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|
10054914 |
Jan 25, 2002 |
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Current U.S.
Class: |
424/490 |
Current CPC
Class: |
A61K 9/12 20130101; A61K
9/0043 20130101; A61K 9/0048 20130101; A61P 17/02 20180101; A61K
9/2081 20130101; A61K 38/193 20130101; A61K 9/1652 20130101; A61K
35/747 20130101; A61K 9/1658 20130101; A23K 20/189 20160501; A61K
38/21 20130101; A61K 35/745 20130101; A61P 37/08 20180101; A61K
9/1623 20130101; A61K 38/20 20130101; A61P 1/12 20180101; A61P
37/02 20180101; A61P 31/12 20180101; A61K 38/4873 20130101; C12N
1/04 20130101; C12N 9/98 20130101; A61P 27/02 20180101; A23K 50/30
20160501; A61P 31/16 20180101; A23K 40/30 20160501; A61K 38/1816
20130101; A61K 38/23 20130101; A61P 11/00 20180101; A61K 38/28
20130101; A61P 35/00 20180101; A61P 1/14 20180101; A61K 9/006
20130101; A61K 35/747 20130101; A61K 2300/00 20130101; A61K 35/745
20130101; A61K 2300/00 20130101; A61K 38/23 20130101; A61K 2300/00
20130101; A61K 38/20 20130101; A61K 2300/00 20130101; A61K 38/193
20130101; A61K 2300/00 20130101; A61K 38/28 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/490 |
International
Class: |
A61K 047/00; A61K
009/16 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 25, 2001 |
AU |
PR2729 |
Claims
1-32. (Cancelled).
33. A method for stabilizing a biologically active material
comprising the steps of: (a) fluidizing microparticles within a
processing chamber to form a fluidized bed of said microparticles,
wherein said microparticles comprise water-soluble gel forming
solid particles; (b) under saturated moisture conditions, spraying
a coating liquid onto said fluidized bed of step (a) from beneath
said fluidized bed to coat said microparticles with said coating
liquid, wherein said coating liquid comprises: (i) at least one
biologically active material, (ii) a sugar polymer, and (iii) a
water soluble/miscible solvent; and (c) drying the resulting coated
microparticles of step (b).
34. The method of claim 33, further comprising coating the
resulting microparticles of step (c) with a coating selected from
the group consisting of an enteric coating, a film coating, a
moisture repellant coating, a taste-masking coating and a
combination of any such coatings.
35. The method of claim 33, wherein the drying in step (c) is heat
drying.
36. The method of claims 33, wherein the biologically active
material is selected from the group consisting of a protein, a
peptide, and a cell.
37. The method of claim 33, wherein the water soluble/miscible
solvent is selected from the group consisting of glycerol,
propylene glycol, and a combination of glycerol and propylene.
38. The method of claim 33, wherein the sugar polymer is selected
from the group comprising dextran, fructose, fruitose, glucose,
invert sugar, lactitol, lactose, maltitol, maltodextrin, maltose,
mannitol, sorbitol, sucrose, trehalose, isomalt, xylitol,
polydextrose, and a combination thereof.
39. The method of claim 33, wherein the water soluble gel forming
solid particles comprise a member selected from the group
consisting of an acrylate, acrylate derivative, albumin, alginate,
carbomer, carrageenan, cellulose, cellulose derivative, dextran,
dextrin, gelatine, polyvinylpyrrolidone, and starch.
40. The method of claim 33, wherein said method is conducted in a
moisture saturated environment.
41. The method of claim 33, wherein said method is conducted in an
oxygen free environment.
42. The method of claim 33, wherein the coated microparticles are
formed into a composition for injection, a sublingual tablet, an
oral tablet, a sustained release sublingual tablet, microcapsules,
pessaries, preconstituted solid dose for nasal spray or drops,
aqueous drops, eye wash or drops, skin washing solutions, or a feed
premix.
43. The method of claim 33, wherein said microparticles have a
particle size of 50 microns to one millimeter.
44. The method of claim 33, wherein the biologically active
material is selected from the group consisting of a hormone,
cytokine, growth factor and a combination of any two or more
thereof.
45. The method of claim 33, wherein the biologically active
material is selected from the group consisting of a human or animal
growth hormone, erythropoietin, calcitonin, interferon,
interleukin, insulin and colony stimulating factor.
46. The method of claim 33, wherein the biologically active
material is an enzyme.
47. The method of claim 46, wherein said enzyme is selected from
the group consisting of streptokinase, muramidase, pancreas,
amylase, protease, lypase, cellulase, bromelain and papain.
48. The method of claim 33, wherein the biologically active
material is glucan.
49. The method of claim 48, wherein said glucan is
.beta.-1,3-glucan.
50. The method of claim 33, wherein the biologically active
material is a microorganism.
51. The method of claim 50, wherein said microorganism is Bifidus
or Lactobacilli.
52. A product produced by the method of any of claims 33 to 51.
Description
FIELD
[0001] The present invention generally relates to a method of
preparing biological materials, and particularly, but not
exclusively, to a method of preparing biological proteins.
BACKGROUND
[0002] Many biological materials, such as proteins or whole cells,
which may be useful in treatment and prevention of human and animal
diseases or as food supplements, for example, are known to have a
limited shelf life. This limitation is generally considered to be a
result of protein instability at storage temperature, for example
room temperature. The shelf life of certain proteins, and/or, cell
cultures, may be extended by storing them at refrigeration
temperatures (that is, 4.degree. C. to 8.degree. C.), however, even
at such temperatures a shelf life of less than eighteen months is
common.
[0003] As will be appreciated, biologically active proteins are
generally folded in a complex three dimensional manner which is
unique to each protein. The proteins are generally organised on
three levels; having a primary structure, consisting of a linear
chain of covalently bonded amino acid residues (a peptide chain); a
secondary structure, in which the peptide chain folds into regular
patterns (such as, a helices and .beta.-pleated sheets); and a
tertiary structure in which the folded chain further folds upon
itself to form a compact structure. In addition, some proteins
consist of more than one polypeptide chain held in close
arrangement to form what is referred to as the quaternary
structure. It is the tertiary and/or the quaternary structure which
dictates a proteins ultimate biological activity.
[0004] The ultimate structure of a protein may be affected by a
number of environmental factors; for example, temperature, pH, the
presence or absence of certain .alpha.-factors or metals, presence
of oxygen, enzymes, oxidising or reducing agents and the presence
of water or moisture. Where conditions are not optimal, a protein
may not form properly or may denature, such that its biological
function is lost, or is at least diminished.
[0005] The cells of animals, plants and microorganisms may be
considered complex protein materials in the broadest sense as they
contain numerous proteins enclosed by a cell membrane and/or cell
wall, which membrane or wall inturn presents additional proteins at
the cell's surface. As with proteins, the viability of a cell is
dependent on the environment in which it resides; for example,
temperature, pH, the presence or absence of certain co-factors or
metals, presence or absence of certain nutrients, metabolic waste,
oxygen, enzymes, oxidising or reducing agents and the presence of
water or degree of moisture may individually or collectively act to
effect viability. As an example, the bacteria Lactobacilli and
Bifidus, which are of commercial significance due to their common
usage in yogurt or as a probiotic in human or animal health
nutritional products, generally can survive at 4.degree. C. for
only a short period of time. At temperatures elevated above
4.degree. C., or during heat/freeze-drying, for example, such
bacteria die due to dehydration. The main cause of the death of the
bacterial cells is thought to be attributed to the denaturation of
the proteins residing within the cell and at the cells surface.
[0006] Cell cultures (including bacterial cell cultures) and
biological proteins are normally made in solution. However, water
is known to hydrolyse protein in a time and temperature dependent
manner resulting in denaturation and potential loss of function.
Dehydrating such cultures or protein solutions may not improve
their stability as during dehydration, and at the high temperatures
at which known dehydration procedures may occur, the proteins may
also be denatured. Refrigeration of cell cultures and proteinaceous
solutions, or the freeze-drying thereof, has been used in an
attempt to curb such problems.
[0007] Freeze-drying under vacuum (lyophilization) is commonly used
in industry to prepare proteins for use in vaccines and the like.
The process traditionally involves freezing a solution of the
biological protein removing ice crystals therefrom by converting
them into water vapour under vacuum (sublimation).
[0008] Unfortunately, this process can cause damage to the native
structure of the protein.
[0009] To help increase the stability of a biological protein being
prepared by freeze-drying, additives such as buffering or
stabilising agents may be used in the product formulation. However,
during freeze-drying, when the temperature of the solution is
slowly reduced to minus 20.degree. C. over a period of days, the
additives may solidify at different freezing points. As a result,
the end product may be a fine puffy cake-like substance actually
made up of different layers, each representing an individual
component. In essence, the additives added to protect the
biological protein may be physically and chemically separated
therefrom rendering them useless as protective agents.
[0010] An alternative procedure, which is commonly used in the food
and dairy industry, to make dry fruit concentrates and milk
powders, for example, is spray-drying-using-heat. This process
involves spraying a fine mist of solution downwards from the top of
a spray tower against an upward current of hot air. The hot air
removes water from the droplets before they reach the bottom of the
tower. Spray drying normally operates at an inlet air temperature
exceeding 190.degree. C. and the product temperature may well
exceed 60.degree. C. At this operating environment, most of the
biological protein or cells, such as bacterial cells, denature.
[0011] Another protein preparation process known in the art is
supercritical fluid drying. In this process, biological agents such
as peptides, proteins and nucleic acids are maintained in an
aqueous solution until particle formation.
[0012] The aqueous solvent is removed at the time of particle
formation using controlled hydrogen-bonding solvents, such as
ethanol, acetone, and isopropanol in carbon dioxide above the
critical point of the supercritical fluid solvent mixture.
[0013] Fluid bed spray drying is a modified spray-drying-using-heat
technology. The process is commonly used in the pharmaceutical and
chemical industry for tablet granulation and/or for drying heat
stable materials. The process involves spraying a fine mist of
solution containing actives downward from the top of a spray head
towards a mass of dry excipients. Simultaneously, an upward current
of hot air is passed through the mass of excipients to create a
fluidized bed. The hot air removes water from the fluidized wet
solids at the bottom of the fluid bed.
[0014] Fluid bed spray drying technology may be applicable to
pharmaceutical proteins which are heat stable around 50.degree. C.
to 60.degree. C. However, the native structure of the protein may
be compromised and accordingly the protein may loose all, or at
least some, of its biological activity.
[0015] Further problems may be associated with fluid bed spray
drying as described above; for example, the spray nozzles, which
are positioned near the top of the processing chamber, are required
to have substantial clearance above the surface of the fluidized
bed of excipient materials so that the such materials do not block
the spraying nozzles; a substantial amount of the coating material,
or liquid containing the active ingredient(s), may block the
nozzles' filter system leading to processing loss; and such top
spraying fluid bed operation may only be ideal for granulation
rather than for spray coating purposes.
[0016] Fluid bed spray drying apparatus have been designed which
spray liquid containing the active ingredient(s) from the bottom of
the processing chamber. For example, the Roto-processor.TM.
(Aeromatic, Switzerland) designed for pellet coating (pellets of
approximately 1 mm or above in diameter), and the Aerocoater.TM.
processor (Aeromatic, Switzerland) designed for coating kernels,
granules, pellets and small tablets. It is considered that neither
the Roto-processor.TM. nor the Aerocoater.TM. are designed for
microparticle coating.
[0017] Of the techniques available, prior to the development of the
present invention, for preparing biological proteins and cells, the
technique of microencapsulation may be considered the most useful.
Typically, no major equipment is required and the batch size can be
as small as 10 g to 20 g thus making it useful for the preparation
of biological proteins that may not be plentiful. This process uses
organic solvents to solubilize the biological protein which is then
encapsulated in polymeric microspheres using either a
water-in-oil-in-water (w/o/w) or a solid-in-oil-in-water (s/o/w)
emulsion method. Protein is captured into the solid microspheres
after water is removed by simple filtration and the solvent is
evaporated off.
[0018] Microencapsulation technology has been used to make carbon
or self-adhesive paper in the paper industry and at least in Japan,
food products, such as artificial fish eggs and decorative products
are made using gelatin microcapsules to entrap fish or meat
flavours.
[0019] While microencapsulation may be considered a favourable
means to prepare biological proteins and whole cells for storage
and future use, the technology is still at the developmental stage
in the pharmaceutical and biotechnological industries. The
technology has apparent difficulties in that proteins are likely to
be denatured by the solvents used and by the necessary
emulsifying/homogenising process. In addition, the quality of a
product produced according to this process, may be considered
undesirable due to the fact that traces of solvent remain in the
core of the microcapsules; the traces of such solvents may hamper
the commercialisation of a product produced using this
technology.
[0020] If biologically active proteins and viable cell cultures
could be prepared such that they were substantially stable at room
temperature, it may increase their shelf life and obviate the need
for refrigeration. At the same time, various alternative drug
delivery methods could be explored, such as conventional oral
delivery, sublingual delivery, nasal delivery, buccal delivery,
occal and even dermal delivery. Such alternative administration
methods may minimize the invasive nature of the commonly used
injection delivery, and create vast commercial opportunities to
fully explore the use of all these molecules.
[0021] Bibliographic details of the publications referred to herein
are collected at the end of the description.
OBJECT
[0022] It is an object of the present invention to provide an
improved method of preparing biological materials, and biological
materials produced therefrom, or at least to provide the public
with a useful choice.
STATEMENT OF INVENTION
[0023] In one broad aspect of the present invention there is
provided a method of preparing products containing
moisture-sensitive materials, including biological materials such
as proteins, peptides or live cells, comprising at least the
steps:
[0024] (i) providing a coating liquid comprising at least one
active, a sugar polymer and a water soluble/miscible solvent;
[0025] (ii) providing a quantity of microparticles comprising at
least water soluble gel forming solid particles;
[0026] (iii) fluidizing said quantity of microparticles within a
processing chamber of a suitable apparatus to form a fluidized bed
of said microparticles;
[0027] (iv) spraying said coating liquid onto said fluidized bed
from beneath the fluidized bed to coat said microparticles
therewith under saturated moisture conditions; and
[0028] (v) allowing coated microparticles to dry.
[0029] The process of the invention may further comprise one or
more additional coating steps which further coat the microparticles
with an enteric coating, a film coating, a moisture repellant
coating or taste masking coating.
[0030] Preferably the coated micropartides are heat dried.
[0031] Preferably, said active comprises proteins, peptides, or
cells.
[0032] The coating liquid of the present invention preferably
comprises additional constituents such as amino acids, proteins,
chelating agents, buffers, preservatives, stabilizers,
antioxidants, lubricants and other additives which may act to
compliment the function of, or stabilize, the active contained
therein.
[0033] Preferably said water soluble/miscible solvent is either or
both of glycerol or propylene glycol.
[0034] Preferably said sugar polymer is selected from one of the
following: dextran, fructose, fruitose, glucose, invert sugar,
lactitol, lactose, maltitol, maltodextrin, maltose, mannitol,
sorbitol, sucrose, trehalose, isomalt, xylitol, polydextrose; or
combination thereof.
[0035] Preferably said water soluble gel forming solid particles
comprise at least one or more of the following; acrylate and
derivatives, albumin, alginates, carbomers, carrageenan, cellulose
and derivatives, dextran, dextrin, gelatin, polyvinylpyrrolidone,
and starch.
[0036] Preferably binding agents selected from one of the following
polymers of acrylate and derivatives, albumin, alginates,
carbomers, carrageenan, cellulose and derivatives, dextran,
dextrin, gelatin, polyvinylpyrrolidone, starch or combination
thereof.
[0037] Preferably the process is conducted in a Huttlin
Turbojet.TM. Coater.
[0038] Preferably the product processing weight exceeds 50% w/v of
the fluid bed processing chamber. More preferably the processing
weight exceeds 75% w/v.
[0039] Preferably the process is conducted in a moisture saturated
environment.
[0040] Preferably the process is conducted within the processing
chamber of the apparatus in an enclosed sterile environment.
[0041] Preferably the process is conducted in an oxygen-free
environment. In such case, the air within the processing chamber
may be replaced by nitrogen, or another suitable inert gas.
[0042] A room temperature stable product produced according to the
method herein described.
[0043] Preferably, said product contains at least one of a protein,
peptide or a cell.
[0044] Preferably said product is suitable for use in a composition
for injection, as sublingual tablets, oral tablets, sustained
release sublingual tablets, microcapsules, feed premix, pessaries,
pre constituted solid dose for nasal spray or drops, aqueous drops,
eye wash or drops, or a skin washing solution.
[0045] A method as herein described when used to stabilize
biological materials.
[0046] A method for creating stable sustained release tablets or
microcapsules for ingestion by an animal, including a human.
[0047] A method for creating a tablet or microcapsules to be
administered to an animal, including a human, said tablet having a
protective enteric coating. Examples of enteric coating materials
which may be used in the invention include cellulose acetate
phthalate, cellulose acetate succinate, cellulose acetate
trimellitate, hydroxypropylmethyl cellulose phthalate,
hydroxypropylmethyl cellulose acetate succinate, polyvinyl acetate
phthalate, methacrylic acid/methyl methacrylate copolymer and
methacrylic acid/ethylacrylate copolymer.
[0048] A method of producing a substantially room temperature
stable antidiarrhoea agent as herein described.
[0049] A method of producing a substantially room temperature
stable growth promotant formulation as herein described.
[0050] A method of producing a substantially room temperature
stable weight loss agent as herein described.
[0051] A method of producing a substantially room temperature
stable tablet or microcapsules containing 13-1,3-glucan as herein
described.
[0052] A method of producing a substantially room temperature
stable product containing erythropoietin (EPO) as herein
described.
[0053] A method of producing a substantially room temperature
stable product containing interferon as herein described.
[0054] A method of producing a substantially room temperature
stable product containing Bifidus.
[0055] A method of producing a substantially room temperature
stable product containing Lactobacilli.
[0056] A method of producing a substantially room temperature
stable product containing Lactobacilli and Bifidus.
[0057] A method of producing a substantially room temperature
stable product containing alternative probiotics or micro
organisms.
[0058] Substantially room temperature stable products produced by
the method described herein.
[0059] A composition comprising a core of microparticles coated
with an active and sugar polymer coating layer.
[0060] Use of compositions as herein described for the delivery of
biological materials to a human or animal.
[0061] The invention may also be said broadly to consist in the
parts, elements and features referred to or indicated in the
specification of the application, individually or collectively, in
any or all combinations of two or more of said parts, elements or
features, and where specific integers are mentioned herein which
have known equivalents in the art to which the invention relates,
such known equivalents are deemed to be incorporated herein as if
individually set forth.
PREFERRED EMBODIMENTS
[0062] These and other aspects of the present invention, which
should be considered in all its novel aspects, will become apparent
from the following description, which is given by way of example
only. It will be appreciated that, while not explicitly mentioned
herein, a number of modifications may be made to the invention
without departing from the scope thereof.
BACKGROUND
[0063] During tablet manufacturing using fluid bed technology the
present inventors discovered that the tablets produced were always
free of bacteria even when the raw materials had bacterial counts
in excess of 1000 CFU/g (CFU=colony forming units). In order to
clarify which of the processing parameters was responsible for the
apparent bactericidal effect, the inventors designed an
experimental protocol which is described in general terms
below.
[0064] The experiment was conducted in a Huttlin Turbojet Fluid Bed
Coater (BWI Huttlin, Daimlerstrasse 7, D-79585, Steinen, Germany)
with a 5 L processing container fitted with three bottom spray
three-component spray nozzle jets and standard 20 micron filter
bags. Samples of Lactobacilli were sourced from Chr. Hansen of 49
Barry Street, Bayswaster, Melbourne, Australia. Lactobacilli with
Bifidus at a ratio of 1:1 was sourced from Gist-Brocade, Australia.
These bacteria are anaerobes which easily perish in the presence of
oxygen. The trial batch size of the fluidized bed was 4 kg, twice
the weight recommended for the 5 L Huttlin Turbojet processing
container used, to ensure the fluidized material was close to the
processing containers filter giving the best chance for the
bacteria to escape through the 20 micron filter. The solid core
(tablet granule core materials) comprised 66% w/w dextrose, 13% w/w
gelatin, 15% w/w starch. The spraying liquid containing bacteria
comprised either 1.16.times.10.sup.12 CFU Lactobacilli, or
3.5.times.10.sup.12 CFU Lactobacilli/Bifidus, with 3% w/w mannitol,
1% w/w albumin, 1% w/w glycerol, 1% w/w sodium phosphate buffers
and made up to 1000 ml with purified water.
[0065] The solid core material was loaded into the Huttlin Turbojet
by vacuum and fluidized at a rate of 250 or 300 cubic meter of
air/hour. Subsequently, the spray liquid was sprayed into the
processing container at a rate of 30 gram/minute. The process was
conducted at a product temperature of 40-45.degree. C. The material
was dried to less than 5% moisture content at a product temperature
of 40.degree. C.
[0066] It was discovered that running the experiment with a
fluidisation rate set at 250 cubic meters of air/hour was not
sufficient. At this rate the fluidized material crashed when 95% of
the liquid was sprayed into the solid core. Fluidisation at a rate
of 300 cubic meters of air/hour overcame this problem.
[0067] Samples of the granules obtained during this process were
retained and the rest of the granules were compressed into tablets
after blended with a standard tablet lubricant. The granules and
the tablets were analysed to assess their live bacterial count. The
results obtained are provided in Table 1 below.
1TABLE 1 Theoretical activity Reported activity in Reported
activity in CFU/4 kg Sample Description in CFU/4 kg time zero CFU/4
kg time zero sample kept at 4.degree. C. for 60 days Chr. Hansen
Granules 1.16 .times. 10.sup.12 1.70 .times. 10.sup.11 1.64 .times.
10.sup.11 Chr. Hansen Tablet 1.12 .times. 10.sup.12 4.40 .times.
10.sup.11 4 .times. 10.sup.10 Gist-Brocade Granules 3.52 .times.
10.sup.12 9.80 .times. 10.sup.10 4.80 .times. 10.sup.10
(Lactobacilli only) (No Bifidus detected) Gist-Brocade Tablet 3.44
.times. 10.sup.12 6.00 .times. 10.sup.10 1.2 .times. 10.sup.10
(Lactobacilli only) (No Bifidus detected)
[0068] It was extremely surprising to detect the presence of viable
bacterial cells in the samples after the treatment the bacterial
cells were exposed to in the processing of the granules and
tablets; heating, possible total ventilation through the 20 micron
filter, mechanical milling, air drying and tablet compression. That
a substantially high number of bacteria survived during these
experiments did not elucidate why the tablets the inventors had
previously produced were bacteria free. The results did, on the
other hand, suggest a novel process to prepare samples or products
containing live bacteria in a stabilized form.
[0069] The suppliers of the lactobacilli and bifidus cultures
indicated that these bacteria are extremely temperature sensitive,
and heat labile. When such cultures are processed using
freeze-drying, bacterial counts are noted to drop by 90-99%.
Further, the suppliers have indicated that the bacteria are also
particularly sensitive to standard tablet compression processes and
accordingly that a further drop in activity of the bacteria of
approximately 99% may be observed.
[0070] Accordingly, the inventors have discovered a novel heat
drying method which appears to be superior to known industrial
methods of preparation, such as freeze-drying. The inventors have
further found that bacteria processed in this novel manner (ie in
micro-capsules) can survive tablet compression pressure around 5 to
8 tons/square inch.
[0071] General Description
[0072] The present invention provides a novel way of drying and
preserving moisture-sensitive materials, particularly biological
materials such as proteins, peptides and plant and animal cells,
including micro-organisms. In general terms, the method combines
the technologies of fluid bed spray processing and
micro-encapsulation.
[0073] In general terms, the process involves the spraying of a
liquid containing at least an active of interest, in combination
with at least one sugar polymer, and a water miscible/soluble
solvent, onto an acceptable particulate excipient material
(microparticles) which is appropriately fluidized in a processing
chamber, at temperatures elevated above room temperature. The
coating of said microparticles provides for the stable
micro-encapsulation of the active ingredient.
[0074] As used herein an "active" generally includes proteins,
peptides and cells. However, those of skill in the art to which the
invention relates will readily appreciate other materials or active
agents which may benefit from preparation according to the
invention. It will be appreciated that as used herein the term
"proteins", "peptides" and "cells" refer to those which have been
produced artificially in the laboratory, via chemical synthesis, or
recombinant techniques, in addition to those which are naturally
occurring.
[0075] The microparticles comprise water soluble gel forming solid
particles, preferably having an adhesive surface, consisting of
either natural or synthetic polymers or monomers which can tolerate
relative high moisture content without turning into liquid or semi
solids. For example, the microparticles preferably comprise at
least one of acrylate and derivatives, albumin (for example egg
albumin (albumen)), alginates, carbomers, carrageenan, cellulose
and derivatives, dextran, dextrin, gelatin, polyvinylpyrrolidone
(for example Povidone), starch or a combination thereof.
Preferably, the microparticles comprise albumin, gelatin, and
pregel starch (for example, pregel maize starch). The
microparticles are preferably 100 microns in diameter, however, it
will be appreciated that alternative sizes may be utilised in the
invention; for example 50 micron to 1 mm particle size. It should
be noted that the water soluble gel forming solid particles of the
microparticles, may be referred to herein as a "hydrogel core".
[0076] It will be appreciated that the composition of the
microparticles used in the invention ensures the liquid spray
material containing the active binds efficiently to the surface of
the microparticles without agglomeration or loss.
[0077] During processing the microparticles are required to be
saturated with moisture to ensure the surface of the particles are
not overheated and a thin film is formed on the surface thereof.
The composition also has the advantage that it dries in a
comparatively slower manner than hard surface particles and also in
a continuous manner to give a complete surface coating.
[0078] As described briefly above, the spray or coating liquid
comprises at least an active, a sugar polymer and a water
soluble/miscible solvent. The sugar polymer is preferably mannitol,
isomalt, xylitol, polydextrose or dextran.
[0079] However, it will be appreciated that alternative polymers
may be used depending on the precise nature of the active contained
within the solution. Suitable sugar polymers may include, for
example, fructose, fruitose, glucose, invert sugar, lactitol,
lactose, maltitol, maltose, maltodextrin, sorbitol, sucrose,
trehalose, or combinations thereof. The water soluble/miscible
solvent is preferably either or both of glycerol or propylene
glycol; however, those of general skill in the art to which the
invention relates may realize alternative solvents suitable for use
in the invention.
[0080] The spray or coating liquid may also contain additional
constituents such as further proteins, amino acids, diluents,
chelating agents, buffers, preservatives, stabilizers,
antioxidants, lubricants and other additives which may act to
compliment the function of, or stabilize, the particular active
contained therein. The precise nature of such additional
constituents, will depend on the nature of the active. However,
examples each include: amino acids, lysine, glycine, L leucine,
isoleucine, arginine, cysteine; proteins, human serum proteins,
albumin (for example egg albumin (albumen)), gelatin; buffers,
various sodium phosphate buffers, citric/citrate buffers, tris
buffer; preservatives, derivatives of hydroxybenzoic acids;
antioxidants, vitamin E, ascorbic acid; lubricants, water miscible
silicone/silicates; chelating agents, citric acid, EDTA, EGTA.
Those of skill in the art will appreciate a variety of other
proteins, amino acids, diluents, chelating agents, buffers,
preservatives, stabilizers, antioxidants and lubricants which may
be suitable for use in the present invention.
[0081] The process is preferably conducted in an enclosed sterile
environment. As used herein a "sterile environment" is taken to be
an environment which is substantially free of contaminating
material. Generally such "contaminating material" will comprise
microorganisms or the like however, those skilled in the art will
appreciate other materials which may be desired not to be present
during processing of a product.
[0082] The environment in which the process is conducted is
preferably free of oxygen to minimise oxidation of actives, for
example. This may be achieved by replacing the air contained within
the processing chamber, in which the processing of the method of
the invention substantially takes place, with an inert gas,
preferably nitrogen. However, it will be appreciated that
alternative gases may be utilised, such as carbon dioxide.
[0083] As briefly mentioned herein before, the microparticles are
required to be saturated with moisture. Accordingly, the process is
said to be conducted in a moisture saturated environment. In a
"moisture saturated environment" the conditions are such that the
surface of the microparticles will begin to dissolve changing from
a totally solid state to a substantially liquid state. Moisture
saturation is achieved in the present invention by over spraying
the coating liquid onto the hydrogel core.
[0084] The process of the invention may be carried out in any
appropriate fluid bed spraying apparatus. In the Examples
elucidated herein a CPU Driven Turbojet.TM. Fluid Bed Coater,
manufactured by BWI Huttlin (Daimlerstrasse 7, D-79585, Steinen,
Germany) has been used. Those of general skill in the art to which
the invention relates will be familiar with such apparatus.
However, further information may be readily obtained from the
manufacturer if necessary.
[0085] It will be appreciated that modifications may be made to the
apparatus used in the process of the invention in order to
facilitate efficient and effective microencapsulation. For example,
the Huttlin Turbojet used in the examples described herein, was
custom modified as follows:
[0086] The spray nozzle was redesigned such that the centre part of
the nozzle (which delivers the liquid spray to the processing
chamber) may be removed during operation of the apparatus, for
cleaning or unblocking the nozzles. This modification allows for
continuous processing.
[0087] The central air return column present in the standard
Huttlin Turbojet apparatus was rearranged and replaced with a
cone-like arrangement such that at high velocity, the fluidized
material moves in a vortex-like manner, and at low velocity,
circulates in a whirlpool motion. It is considered that such a
modification allows for improved coating of the micro
particles.
[0088] All contact surfaces were extra mirror polished such that
they can be readily heat sterilised after the standard
Cleaning-In-Place cycle.
[0089] Additional compressed air spray nozzles facing the inner
surface of the processing container/chamber wall were added
surrounding the existing dynamic filter system arrangement. This
modification may provide a continuous stream of compressed air
flowing from the top of the chamber along the surface of the inner
chamber ensuring the working surface in the process container is
cleaned continuously. This modification ensures the equipment can
be operated continuously without repeat cleaning.
[0090] The processing air is so designed that at any stage a
recirculating inert gas, such as nitrogen, can be introduced for
fluidisation instead of air. This modification may reduce
biological protein oxidation and increase anaerobic bacteria
stability.
[0091] The microencapsulation process of the invention uses an
unconventional bottom spray coating operation. That is, spraying of
the spray or coating liquid occurs from the bottom of the
processing container upwards. As such, it will be appreciated that
the spray nozzles are actually embedded within the fluidized bed.
Depending on batch size there can be up to thirty eight spraying
nozzles operating at the same time.
[0092] The spray liquid is processed in such manner that it
transforms itself into a continuous glassy film ("bioglass" film)
wrapped around the solid surface of the fluid bed particles. The
transformation from liquid to glassy solid is rapid, preventing
denaturing of the biological protein or microorganism. The active,
such as a biological proteins or micro organisms do not suffer in
the heat, which is dissipated by the latent heat of evaporation of
water.
[0093] The process of the present invention preferably involves the
over weighting of the microparticles into the processing chamber.
In normal fluid bed operation it is recommended by equipment
manufacturers not to exceed 50% w/v capacity of the processing
chamber. For example, if the processing container is 100 L,
processing material weight should not be more than 50 kg.
[0094] However, the process of the present invention allows for
(and it is preferable to do so) the processing weight:container
volume to be more than 50% w/v.
[0095] In this manner, the weight of the microparticles may act
like a sieve so that when the encapsulation process is initiated,
the spray liquid will not pass through the fluidized microparticles
and out through the dynamic filter system resident at the top of
the processing chamber.
[0096] The method of the invention may be described in general
terms as follows:
[0097] 1 Solid hydrogel particles (microparticles) of a suitable
constitution are loaded into the Turbojet by vacuum and fluidized.
Fluidization may occur at a rate of between 200 to 500 cubic meters
per hour.
[0098] 2 The microparticles are preferably heated to 30.degree. C.
to 80.degree. C., more preferably to 60.degree. C., for
approximately one hour with high velocity processing air so they
are fluidized in a vortex-like motion, ensuring that the inner part
of the microparticles are dry.
[0099] 3 Microparticle temperature is preferably reduced to
35.degree. C. to 55.degree. C. and the processing air velocity is
similarly preferably reduced so that the microparticles move in a
whirlpool-type manner.
[0100] 4 When the micro-particle temperature reaches preferably
approximately 40.degree. C. to 50.degree. C., the processing air is
preferably replaced with an inert gas, such as nitrogen. This step
is preferably held for at least approximately 10 minutes to ensure
all the air is replaced with nitrogen.
[0101] 5 Active is immobilised in an appropriate spray or coating
solution. The base solution is preferably heated to 38.degree. C.
to allow complete solid dissolution. Prior to Turbojet spray
coating the biological materials are added to the base solution
(mixing at approximately 60 rpm) and mixed well.
[0102] 6 A desired quantity of coating solution is then Turbojet
Spray Coated onto the fluidized micropartides preferably at high
speed (preferably at the highest available speed) so the
microparticles are saturated with moisture but still able to freely
flow in a whirlpool manner. Spray coating preferably takes place at
a rate of 30 grams to 60 grams per minute. The spraying of said
coating solution or liquid onto the microparticles occurs from
beneath the fluidized bed.
[0103] 7 Turbojet coating speed is slowed to preferably between 10
grams to 20 grams per minute when the microparticles are saturated
with moisture, to ensure the bed of microparticles is continuously
flowing in a whirlpool manner. In this manner the coating solution
containing the biological protein is continuously dehydrated in a
moisture free nitrogen environment, for example. The product is
typically dried to result in a water activity of less than
0.25.
[0104] It will be appreciated that the above processing steps and
parameters may be altered to accommodate the production of various
product forms, or products comprising different actives.
Alterations may be made for example to: the inlet process air
temperature, the product temperature, fluidized air volume, liquid
spraying speed, spray liquid temperature, spray liquid viscosity,
spray liquid solid content, total core surface area, water
solubility of core, humidity of inlet air, compressed air spraying
pressure, the apparatus filter pore size, and the frequency of auto
dedusting. Where an alteration is made to one parameter, a person
of general skill in the art to which the invention relates will
readily be able to identify any corresponding adjustments which may
be required in another parameter to compensate for the first said
alteration. In addition, by increasing the molecular weight of the
hydrogel core sustained release solid dosage can be created.
[0105] Further, additional coating steps may be added to the above
general process according to the invention, in order to obtain
products having desired characteristics. For example, prior to or
after the drying step, the resultant product, or, microcapsules,
may be coated with further coatings. Those of skill in the art to
which the invention relates will immediately realize situations
where this may be advantageous; for example, where a resultant
product is desired to be administered orally, enteric coatings
which may protect the product from degradation in the stomach,
and/or, those which allow for sustained or slow release of the
active therefrom may be utilized. Generally such further coating
will be carried out at a similar coating rate as that used for
coating the microparticles with the initial coating liquid.
[0106] The batch size for processing may vary according to the
volume of the processing chamber of the apparatus used, and whether
overloading thereof is required. In the Examples which are
described herein, the batch size is typically 4 kg. "Batch size"
refers to the total solids used in the processing of the product
and constitutes solids contained in both the microparticles,
coating solution, and any additional coating solutions used to
formulate the product. Accordingly, as used herein percentages of
particular constituents are expressed in terms of the percentage of
the total batch size.
[0107] Various other modifications will become apparent from the
Examples provided herein.
[0108] The invention is now further elucidated by reference to the
following specific non limiting examples, and figures.
[0109] In the figures:
[0110] FIG. 1: Sublingual delivery of EPO in rats. Reticulocyte
counts show it as 10.sup.10/L measured from withdrawn blood samples
are plotted against days on or after treatment.
[0111] The normal range is depicted by (two thick lines);
[0112] treatment groups were by subcutaneous injections (line with
diamond in the middle) of 50 IU on day one;
[0113] sublingual deliver of 125 IU EPO (line with square in the
middle) on days one, two and three;
[0114] sublingual delivery of 125 IU EPO (a line with light
coloured triangle delivered on days one, two, three, four and
five.
[0115] FIG. 2: Shows stability of EPO tablets over nine months at
4.degree. C. (a line with a diamond through it) and a room
temperature (a line with a square through it).
EXAMPLES
Example 1
Stabilization of Microorganisms
Example 1A
[0116] Eight litres of live Bifidus culture was obtained from an
original 3000 L liquid culture from Sine Pharmaceutical Co., Ltd. #
905, Xinjinqiao Rd., Pudong, Shanghai, P. R. China. Data supplied
from the manufacturer established that 3000 L of fermentation
liquid contains a total of 3.times.10.sup.16 CFU (colony forming
units) and yields approximately 8.3 kg of freeze dry material
containing a total of 2.16.times.10.sup.14 CFU of Bifidus; that is,
after freeze drying, there is reduction of approximately 99% live
bacteria population.
[0117] After arrival in the laboratory a sample of the culture was
tested for stability at 4.degree. C. At time zero
2.43.times.10.sup.16 CFU/3000L was recorded. After storage for
fourteen days under 4.degree. C., the count was reduced to
1.5.times.10.sup.14 CFU/3000L; that is, about one in 1500 cells
survived after two weeks storage at 4.degree. C.
[0118] The liquid culture, containing various sugar additives (as
described below) was processed according to the invention in the
following manner:
[0119] 1 Solid micro-particle core (hydrogel core) material was
loaded into the Huttlin Turbojet by vacuum.
[0120] 2 The microparticles were fluidized and heated to 60.degree.
C. for one hour.
[0121] 3 The micro-particle core temperature was reduced to 400 to
45.degree. C.
[0122] 4 The process air was replaced with nitrogen and flushed for
ten minutes.
[0123] 5 Microparticles fluidized at a rate of 300 cubic metres of
air/hour.
[0124] 6 Bifidus coating liquid was turbojet coated onto the
hydrogel core particles under saturated moisture conditions at a
rate of 30 gram/minute.
[0125] 7 Resultant product dried to less than 0.25 water
activity.
[0126] 8 Samples of coated microcapsules were tested as time zero
and after storage at 4.degree. C., 25.degree. C. and 40.degree.
C.
[0127] This bioencapsulation process was conducted using four
different combinations of micro-particle, or hydrogel core, and
coating liquid formulations as indicated below.
2 4 kg Batch SINE RX1 Hydrogel Core 1 Dextrose 2.64 kg Gelatin 0.52
kg Starch 0.60 kg Coating Liquid 1 1 .times. 10.sup.10 CFU Bifidus
Mannitol 0.20 kg Sodium phosphate buffer 0.04 kg Purified water to
1.00 kg SINE RX2 Hydrogel Core 2 Dextrose 2.44 kg Gelatin 0.52 kg
Starch 0.60 kg Egg Albumin 0.20 kg Coating Liquid 2 1 .times.
10.sup.10 CFU Bifidus Mannitol 0.20 kg Sodium phosphate buffer 0.04
kg Purified water to 1.00 kg SINE RX3 Hydrogel core 3 Dextrose 2.44
kg Gelatin 0.52 kg Starch 0.60 kg Egg Albumin 0.20 kg Coatinq
Liquid 3 1 .times. 10.sup.10 CFU Bifidus Dextran 0.20 kg Sodium
phosphate buffer 0.04 kg Purified Water to 1.00 kg SINE RX4
Hydrogel core 4 Dextrose 2.44 kg Gelatin 0.52 kg Starch 0.60 kg Egg
Albumin 0.20 kg Coatinq Liquid 4 1 .times. 10.sup.10 CFU Bifidus
Egg Albumin 0.20 kg Sodium phosphate buffer 0.04 kg Purified Water
to 1.00 kg
[0128] Results recorded are listed in Table 2 below and expressed
as CFU equivalent to 3000-L original concentration.
3TABLE 2 Time zero Time zero Four weeks Formulation PRE Bio- POST
Bio- POST Bio- Code encapsulation encapsulation encapsulation SINE
RX1 at 4.degree. C. 1.5 .times. 10.sup.14 6.00 .times. 10.sup.12
6.00 .times. 10.sup.13 SINE RX2 at 4.degree. C. 1.5 .times.
10.sup.14 2.70 .times. 10.sup.12 3.69 .times. 10.sup.13 SINE RX3 at
4.degree. C. 1.5 .times. 10.sup.14 2.70 .times. 10.sup.12 9.90
.times. 10.sup.12 SINE RX4 at 4.degree. C. 1.5 .times. 10.sup.14
1.41 .times. 10.sup.13 1.29 .times. 10.sup.12 Average 2.99 .times.
10.sup.13 SINE RX1 at 25.degree. C. 1.5 .times. 10.sup.14 6.00
.times. 10.sup.12 1.38 .times. 10.sup.13 SINE RX2 at 25.degree. C.
1.5 .times. 10.sup.14 2.70 .times. 10.sup.12 2.04 .times. 10.sup.13
SINE RX3 at 25.degree. C. 1.5 .times. 10.sup.14 2.70 .times.
10.sup.12 6.30 .times. 10.sup.11 SINE RX4 at 25.degree. C. 1.5
.times. 10.sup.14 1.41 .times. 10.sup.13 6.00 .times. 10.sup.12
Average 1.02 .times. 10.sup.13 SINE RX1 at 40.degree. C. 1.5
.times. 10.sup.14 6.00 .times. 10.sup.12 < 10000 SINE RX2 at
40.degree. C. 1.5 .times. 10.sup.14 2.70 .times. 10.sup.12 4.5
.times. 10.sup.11 SINE RX3 at 40.degree. C. 1.5 .times. 10.sup.14
2.70 .times. 10.sup.12 < 10000 SINE RX4 at 40.degree. C. 1.5
.times. 10.sup.14 1.41 .times. 10.sup.13 < 10000
[0129] The results indicate ten times more live Bifidus survive the
processing according to the present invention compared to
conventional freeze-drying processes. The reaction referred to as
SINE RX2 gave the best stability results.
[0130] It was found that during processing of Sine RX1 moisture was
not effectively picked up by the dextrose contained within the
hydrogel core. When albumin was added to the hydrogel core
formulation (see RX2 to RX4) processing was satisfactory.
Example 1B
[0131] Further batches of Bifidobacterium bifidum 6-1 were imported
from Sine Pharmaceutical Co., Ltd. # 905, Xinjinqiao Rd., Pudong,
Shanghai, P. R. China. The culture used in Example 1A was thought
to contain some waste material which may contribute to instability
of the bacteria in the final bioencapsulated solid micro capsules.
Accordingly, the culture used in the present example had all waste
material removed, was concentrated and resuspended in buffer
solutions. The culture was assayed on arrival from the manufacturer
and a sample was also assayed just prior to use. Results indicated
a bacterial count of 4.1.times.10.sup.8CFU/L.
4 4 kg Batch SINE RX6 Hydrogel core 6 Pregel Maize Starch 3.10 kg
Egg Albumin 0.40 kg Coating Liquid 6 Bifidus 8.2 .times. 10.sup.8
CFU Mannitol 0.10 kg Glycerol 0.30 kg Sodium Alginate 0.06 kg
Sodium phosphate buffer 0.04 kg Purified water to 1.00 kg. SINE RX7
Hydrogel core 7 Pregel Maize Starch 2.44 kg Gelatin 0.52 kg Starch
0.60 kg Egg Albumin 0.20 kg Coatinq Liquid 7 Bifidus 4.2 .times.
10.sup.8 CFU Mannitol 0.05 kg Glycerol 0.15 kg Sodium Alginate 0.06
kg Sodium phosphate buffer 0.04 kg Purified water to 1.00 kg SINE
RX8 Hydrogel core 8 Pregel Maize Starch 2.54 kg Gelatin 0.52 kg
Starch 0.60 kg Egg Albumin 0.20 kg Coatinq Liquid 8 Bifidus 4.2
.times. 10.sup.8 CFU Mannitol 0.025 kg Glycerol 0.075 kg Sodium
phosphate buffer 0.040 kg Purified water to 1.00 kg. SINE RX9
Hydrogel core 9 Pregel Maize Starch 2.52 kg Gelatin 0.52 kg Starch
0.60 kg Egg Albumin 0.20 kg Coating Liquid 9 Bifidus 4.2 .times.
10.sup.8 CFU Mannitol 0.025 kg Glycerol 0.075 kg Sodium Alginate
0.020 kg Sodium phosphate buffer 0.040 kg Purified water to 1.00
kg.
[0132] Processing was carried out according to the protocol used in
Example 1A.
[0133] Results recorded are listed in Table 3 below and expressed
as CFU in 4 kg of microcapsules.
5TABLE 3 Time zero Time zero Two months Formulation PRE Bio- POST
Bio- POST Bio- Code encapsulation encapsulation encapsulation SINE
RX6 at 4.degree. C. 8.4 .times. 10.sup.8 1.56 .times. 10.sup.8 2.24
.times. 10.sup.8 SINE RX7 at 4.degree. C. 4.2 .times. 10.sup.8 5.32
.times. 10.sup.7 6.00 .times. 10.sup.7 SINE RX8 at 4.degree. C. 4.2
.times. 10.sup.8 4.72 .times. 10.sup.7 4.04 .times. 10.sup.7 SINE
RX9 at 4.degree. C. 4.2 .times. 10.sup.8 4.48 .times. 10.sup.7 2.36
.times. 10.sup.7 SINE RX6 at 25.degree. C. 8.4 .times. 10.sup.8
1.56 .times. 10.sup.8 1.64 .times. 10.sup.8 SINE RX7 at 25.degree.
C. 4.2 .times. 10.sup.8 5.32 .times. 10.sup.7 1.56 .times. 10.sup.7
SINE RX8 at 25.degree. C. 4.2 .times. 10.sup.8 4.72 .times.
10.sup.7 2.76 .times. 10.sup.7 SINE RX9 at 25.degree. C. 4.2
.times. 10.sup.8 4.48 .times. 10.sup.7 1.40 .times. 10.sup.7 SINE
RX6 at 40.degree. C. 8.4 .times. 10.sup.8 1.56 .times. 10.sup.8
<100 SINE RX7 at 40.degree. C. 4.2 .times. 10.sup.8 5.32 .times.
10.sup.7 <100 SINE RX8 at 40.degree. C. 4.2 .times. 10.sup.8
4.72 .times. 10.sup.7 <100 SINE RX9 at 40.degree. C. 4.2 .times.
10.sup.8 4.48 .times. 10.sup.7 <100
[0134] It is apparent from the results that the addition of
glycerol to the hydrogel core particles further enhances the
biological stability of Bifidus. It was observed that the addition
of alginate to the coating liquid improved fluidisation but did not
significantly affect the stability of the bacterium in the final
product.
[0135] The results obtained from this example again demonstrate
that the process of the invention may be considered superior to
that of currently used processing techniques, for example
freeze-drying; in SINE RX6 it is seen that 1 in 5 bacteria survived
processing according to the invention as compared to a reported 1
in 100 in the traditional freeze-drying method.
Example 2
Stabilization of Enzymes
[0136] Enzymes are biological proteins which have applications in a
variety of industries; for example, they are used in food
processing, as animal feed additives, and as human and animal
medications.
Example 2a
Stabilized Enzymes Incorporated into Feed as Growth Promotant
[0137] (Enzyme Growth Promotant 2).
[0138] Enzymes such as proteases, lipases, amylases, and
cellulases, for example, are common additives to animal feed. These
enzymes help to increase the bioavailability of the feed.
[0139] Animal feed is often manufactured such that the enzymes,
together with vitamins and minerals such as copper sulfate and
iron, are mixed into the feed. The feed is then generally
palletised by steam injection and extrusion. The operating
temperature of the feed during palletisation can reach 80.degree.
C. and above for approximately ten minutes. Under such conditions
many of the enzymes added to the mix may be denatured. In addition,
when such feed enters the stomach of an animal, many of the enzymes
may be denatured due to the acidic environment therein.
[0140] To counter the loss of enzymatic activity due to feed
palletisation and the acidic environment in the stomach, current
practice is to add massive quantities of enzymes into the feed
premix in the hope that at least some of the enzymes will
survive.
[0141] Accordingly while the use of enzymes in animal feed is
theoretically beneficial, the efficacy has not been consistently
demonstrated to be economically viable.
[0142] Where the added enzymes are sufficiently protected from the
harmful environmental factors to which they may be exposed, there
may be significant economic and growth benefits. Australian Patent
Application AU07872187 describes a growth promotant comprising
microgranules having a core, consisting of one or more immobilised
enzymes, encapsulated within a water soluble film and coated with a
protective enteric coating. Such a product may help overcome the
problems associated with degradation of feed enzymes.
[0143] AU07872187 describes a method of producing such a product
described in the previous paragraph which typically involves
freeze-drying and milling. The present example demonstrates that
the method of the present invention may be used to produce an
equivalent product, which may significantly reduce the cost of
production.
[0144] The formulation for the enzyme growth promotant 2 is as
follows, expressed in terms of a 4 kg batch size (% w/w):
[0145] Hydrogel core: Pregel Maize Starch 67.5%,
polyvinylpyrrolidone (Povidone) 10%.
[0146] Coating liquid: Protease 2.times.10.sup.5 Vitapharm Protease
Units, Amylase 4.3.times.10.sup.5 Vitapharm Amylase Units, Lipase
50 Vitapharm Lipase Units, Cellulase 2.times.10.sup.4 Vitapharm
Cellulase Units, mannitol 2.5%, glycerol 7.5%, polyvinylpyrrolidone
(Povidone) 1.5%, sodium phosphate buffer to pH 7 1%, purified water
to 1 kg.
[0147] Enteric Coating
[0148] Solution (1L batch): Cellulose acetate phthalate 10%, sodium
hydroxide qs to pH 6, water purified to 100%.
[0149] Final Acid
[0150] Rinse Solution: Citric acid qs to pH 3, purified water to 1
L.
[0151] The growth promotant of the present example is preferably
used at a rate of 1 kg/ton feed.
[0152] The growth promotant formulation was processed according to
the invention using the following protocol:
[0153] 1 Hydrogel core material is vacuum loaded into the Huttlin
Turbojet chamber, fluidized and heated to 60.degree. C. for one
hour.
[0154] 2 Hydrogel core product temperature reduced to 45.degree.
C.
[0155] 3 The content of the chamber is fluidized at a rate of 300
cubic metres per hour.
[0156] 4 Coating liquid turbojet coated onto the hydrogel core
under saturated moisture conditions at a rate of 30 g/minute.
[0157] 5 Product dried to less than 5% moisture content.
[0158] 6 Enteric coating solution turbojet coated onto the core at
a rate of 30 g per minute.
[0159] 7 Citric acid solution turbojet coated onto the core at a
rate of 30 g per minute. The acid acts to reconvert the sodium
cellulose acetate phthalate back to cellulose acetate phthalate
offering the enzymes enteric protection through the stomach.
[0160] It is observed that the process of the invention requires
one tenth of the processing time compared with the previous method
of processing described in AU07872187, and also reduces production
costs by up to 50%.
[0161] In addition, it is noted and was observed that the process
does not use any organic solvents or aldehydes, the entire
production can be performed in an enclosed environment in one step,
exposure of operators to enzymes is greatly reduced and only half
the cellulose acetate phthalate is required to offer the same
enteric protection.
[0162] Furthermore, the process resulted in a product which is
stable at room temperature for at least two years.
[0163] The formulation and process of the present example may be
modified by providing an additional final 5% w/w wax coating, such
as low melting point macrogol or PEG, for example. In this case, it
is believed the microcapsules may be incorporated into a feed mix
prior to pelletization, with minimal, if any, disruption to enzyme
structure; the additional wax coating is able to withstand a short
burst of steam and accordingly take up the majority of the heat
used during pelletization.
Example 2b
Stabilized Enzymes as Weight Loss Supplement
[0164] During studies conducted to determine the appropriate dose
rate of the growth promotant 2 described above, it was observed
that dosing at 1 kg/ton feed gives the optimum feed conversion.
However, where the dose rate is increased and reaches 10 kg/ton
feed, the growth promotant formulation is noted to induce
significant weight loss.
[0165] Accordingly, a weight loss enzyme supplement was formulated
and two open trials were conducted in humans.
6 The weight loss supplement comprised: 4 kg Batch Hydrogel core
Pregel Maize Starch 2.70 kg Polyvinylpyrrolidone (Povidone) 0.40 kg
Coating Liquid Protease 2 .times. 10.sup.6 Vitapharm Protease Units
Amylase 4.3 .times. 10.sup.7 Vitapharm Amylase Units Lipase 500
Vitapharm Lipase Units Cellulase 2 X 10.sup.5 Vitapharm Cellulase
Units Mannitol 0.10 kg Glycerol 0.30 kg Polyvinylpyrrolidone
(Povidone) 0.06 kg Sodium phosphate 0.04 kg buffers to pH 7
Purified water to 1.00 kg Enteric Coatinq Solution Cellulose
Acetate Phthalate 0.40 kg Sodium Hydroxide qs to pH 6 Water
purified to 4 kg Final Acid Rinse Solution Citric Acid qs to pH 3
Purified water to 1.00 kg
[0166] The weight loss supplement was processed according to the
invention using the protocol used for the preparation of the enzyme
growth promotant 2, described above.
[0167] Following processing, the microcapsules were packed in
moisture proof sachets, in lots of 1 g. A dosage of one sachet
mixed in water was taken before each meal.
Example 2b(i)
Weight Loss Study 1
[0168] Nine volunteers were recruited to determine whether the
composition has any weight loss effect, when taken as indicated
above.
[0169] Results are given in Table 4 below:
7TABLE 4 Body weight in kg Body Weight in kg Subject Week 0 End of
Six weeks 1 125 116.4 2 107 101.5 3 105 97 4 100 97 5 79 69 6 78 72
7 73.5 66.3 8 73 69 9 68.3 61.5 Total 808.8 746.7 Mean 90 83
[0170] A mean weight loss per person of approximately 1.08 kg per
week, or 7 kg over six weeks, was observed.
[0171] Weight Loss Study 2
[0172] A second study of twenty volunteers was conducted under the
supervision of a medical practioner. Dosage rates were as for
"Weight loss study 1".
[0173] The results of this study are collected in Table 5
below.
8TABLE 5 Body weight in kg Body Weight in kg Subject Week 0 End of
three weeks 1 128 120 2 126 121 3 115 109 4 108 100 5 102 101 6 102
97 7 97 92 8 94 93 9 92 90 10 92 88 11 89 85 12 85 84 13 83 81 14
82 78 15 78 76.5 16 77 70 17 77 70 18 74 71 19 73 72 20 68 60 Total
1842 1758.5 Mean 92.1 88
[0174] Mean weight loss per person was approximately 1.37 kg per
week, or 4.1 kg over three weeks.
[0175] The results obtained from the two isolated studies described
indicated that the enzyme formulation described herein may be an
effective weight loss supplement.
Example 2c
Stabilized Bromelain as Anti-Diarrhoea Medication
[0176] It has been demonstrated that proteases can be used for
treatment of intestinal pathogens in animals, including humans;
AU07858587 and AU02367392. Compositons for delivery of such
proteases have been described comprising: (i) granules comprising a
biologically active material in association with a weak base and
partially coated with a delayed release material soluble in
intestinal juice; (ii) an acidifying agent having a pH between 1.6
to 6; and (iii) a gel forming agent. The resulted preparations are
able to modify the host intestinal surface so that it is not
susceptible to bacterial colonisation. Accordingly, the preparation
is useful for prevention and treatment of diarrhoea.
[0177] The example elucidated below provides an improved method of
manufacturing such an antidiarrhoea formulation.
9 The antidiarrhoea formulation comprised: 4 kg Batch Hydrogel core
Pregel Maize Starch 2.914 kg Polyvinylpyrrolidone (Povidone) 0.40
kg Coating Liquid Bromelain 0.053 kg Cysteine 0.053 kg Mannitol
0.10 kg Glycerol 0.30 kg Polyvinylpyrrolidone (Povidone) 0.06 kg
Sodium phosphate buffers to pH 7 0.04 kg Purified water to 1.00 kg
Enteric Coatinq Solution Cellulose Acetate Phthalate 0.08 kg Sodium
Hydroxide qs to pH 6 Purified water to 8 kg of total batch size
Final Acid Rinse Solution Citric Acid qs to pH 3 Purified water to
1.00 kg
[0178] The antidiarrhoea formulation was processed according to the
invention using the following protocol:
[0179] 1 Hydrogel core loaded into the Huttlin Turbojet processing
chamber by vacuum, fluidized and heated up to 60.degree. C. for one
hour.
[0180] 2 Hydrogel core product temperature reduced to 45.degree.
C.
[0181] 3 Hydrogel bed fluidized at a rate of 300 cubic metres per
hour.
[0182] 4 Hydrogel core turbojet coated with coating liquid under
saturated moisture conditions at 30 g/minute.
[0183] 5 Product dried to less than 5% moisture content.
[0184] 6 Enteric coating solution turbojet coated onto core at a
rate of 30 g/minute.
[0185] 7 Citric acid solution turbojet coated onto the core at a
rate of 30 g/minute; the acid reconverting the sodium cellulose
acetate phthalate to cellulose acetate phthalate providing enteric
protection for the enzymes within the formulation.
[0186] It was observed that this process requires only one twelfth
to the processing time compared with conventional methods used to
produce such a product.
[0187] Further, one twentieth of the amount bromelain is required
in the formulation (due to increased stability of bromelain within
the bioglass matrix).
[0188] In addition, it is noted that the entire production can be
performed in an enclosed environment in one step, exposure of
operators to enzymes is greatly reduced and only one fifth the
cellulose acetate phthalate is required to offer the same enteric
protection as that gained from the known product described
above.
[0189] Overall, the production cost is estimated to be reduced to
one fifth of that where conventional methods are used to create an
equivalent antidiarrhoea formulation.
[0190] Furthermore, it was observed that the process resulted in a
product which is stable at room temperature for at least two
years.
[0191] Example 2c(i): Animal studies involving antidiarrhoea
formulation A study of the efficacy of the formulation produced
according to Example 2c as an antidiarrhoea treatment was conducted
at the Animal Husbandry Research Institute, Jinin Province, China.
Ninety new born piglets of approximately same weight and age were
used in the study.
[0192] The piglets were randomly divided into two groups, equal in
sex, weight and age. One group was designated for treatment with a
Bromelain Preparation according to Example 2c and the other half
were used as a control group. 0.75 g of the Bromelain Preparation
(Example 2c) was mixed with 8.5 g of water into a paste on the day
of use. A first dose of 10 ml was given to the treatment group at
day seven after birth and repeated at day ten. The control group
received no treatment. Observation of diarrhoea incidence was
recorded up to day forty five.
[0193] Results are collected in Table 6:
10 TABLE 6 Treatment Group Control Number of animals 45 45 Birth
weight (kg) average 1.40 1.35 Vaccination against diarrhoea Yes Yes
Weaning weight (kg) average 11.80 11.30 Total weight gained 10.40
9.95 Daily weight gained 249 g 237 g Feed consumed 13 kg 12.7 kg
Feed conversion 1.25 1.29 Pigs that has diarrhoea 3 8 Diarrhoea
incidence (%) 6.7 17.8 Animal alive (%) 100 100 Diarrhoea days 3 5
Other medication used 6 10
[0194] All piglets used in this trial were vaccinated against
diarrhoea. Nonetheless, the treatment group, after two doses of the
bromelain preparation, showed significant weaning weight gain (0.5
kg), reduced diarrhoea incidence (6.7 versus 17.8), reduced
severity (three days versus five days) and reduced number of other
medications used (6 versus 10) compared to control group.
Example 2d
Stabilized Enzyme Bromelain Plus .beta. glucan (B&B
Preparation) as anti-diarrhoea medication
[0195] Addition of .beta.-1,3-glucan to the Bromelain Preparation
of Example 2c potentates the antidiarrhoea action of bromelain.
[0196] B-1,3-glucan, is considered an effective natural
non-specific immunostimulant with free-radical scavenging
properties. It is thought to act by activating macrophages, which
play an essential and pivotal role in the initiation and
maintenance of the immune response in animals, including
humans.
[0197] B-1,3-glucan is known to be orally effective, completely
safe and non-toxic. There are several different types of .beta.
glucan with different levels of activity, the majority of which are
inert and used as simple food fillers. B-1,3-glucan, is however the
most active .beta. glucan, and may be obtained from the cell wall
of yeast.
[0198] B-1,3-glucan may be considered useful in the treatment of
many immune-related indications, such as stress-related
immunosuppression, for example, has been shown to act
synergistically with antibiotics and antiviral medications and to
exhibit antifungal properties. Accordingly, .beta.3-1,3-glucan may
be considered a suitable adjuvant for an improved life-style. Those
of general skill in the art to which this invention relates will
readily be able to identify animal indications which may benefit
from the administration of .beta.3-1,3-glucan.
[0199] The B&B Preparation of the present example comprises, in
the context of an 8 kg batch size:
11 4 kg Batch Hydroqel core for Bromelain Pregel Maize Starch 2.861
kg Polyvinylpyrrolidone (Povidone) 0.40 kg Coating Liquid Bromelain
0.106 kg Cysteine 0.106 kg Mannitol 0.100 kg Glycerol 0.300 kg
Polyvinylpyrrolidone (Povidone) 0.060 kg Standard sodium phosphate
buffer to pH 7 0.040 kg Purified water to 1.00 kg. Hydroqel core
for B-1,3-glucan Gelatin 2.861 kg Egg Albumin 0.400 kg Coating
Liquid B-1,3-glucan 0.106 kg Mannitol 0.100 kg Glycerol 0.300 kg
Polyvinylpyrrolidone (Povidone) 0.060 kg Standard sodium phosphate
buffer to pH 7 0.040 kg Purified water to 1.00 kg Enteric Coating
Solution Cellulose Acetate Phthalate 2.00 0.16 kg Sodium Hydroxide
qs to pH 6 Purified water to 8 kg of total batch size Final Acid
Rinse Solution Citric Acid qs to pH 3 Purified water to 2.00 kg
[0200] The bromelain microcapsules were processed according to the
invention using the following protocol:
[0201] 1 Hydrogel core for bromelain loaded into the Huttlin
Turbojet chamber by vacuum, fluidized and heated up to 60.degree.
C. for one hour.
[0202] 2 Hydrogel core product temperature reduced to 45.degree.
C.
[0203] 3 Hydrogel bed fluidized at a rate of 300 cubic metres per
hour.
[0204] 4 Coating liquid turbojet coated onto the hydrogel core
under saturated moisture conditions at a rate of 30 g/minute.
[0205] 5 Product dried to less than 5% moisture content.
[0206] 6 Sodium cellulose acetate phthalate solution turbojet
coated onto the core product at a rate of 30 g/minute.
[0207] 7 Citric acid solution turbojet coated onto the core as a
final coat at 30 g/minute; the acid acts to convert the sodium
cellulose acetate phthalate to cellulose acetate phthalate
providing enteric protection for the enzymes within the
formulation.
[0208] The .beta.-1,3-glucan microcapsules were processed according
to the invention using the following protocol:
[0209] 1 Hydrogel core material for .beta.-1,3 glucan loaded into
the Huttlin Turbojet chamber by vacuum, fluidized and heated up to
60.degree. C. for one hour.
[0210] 2 Hydrogel core product temperature reduced to 45.degree.
C.
[0211] 3 Process air replaced with nitrogen and flushed for ten
minutes.
[0212] 4 Hydrogel bed fluidized at a rate of 300 cubic metres per
hour.
[0213] 5 .beta.-1,3 glucan coating turbojet coated onto the
hydrogel core under saturated moisture conditions at a rate of 30
g/minute.
[0214] 6 Resultant product dried to less than 5% moisture
content.
[0215] 7 Sodium cellulose acetate phthalate solution turbojet
coated onto the core product at a rate of 30 g/minute.
[0216] 8 Citric acid solution turbojet coated onto the core as a
final coat at a rate of 30 g/minute; the acid acts to convert the
sodium cellulose acetate phthalate to cellulose acetate phthalate
providing enteric protection for the enzymes within the
formulation.
[0217] The B&B formulation is then prepared by mixing 4 kg of
the bromelain microcapsules with 4 kg of the .beta.-1,3 glucan
microcapsules.
[0218] Using this protocol, each 750 mg of B&B Preparation
contains 10 mg bromelain and 10 mg .beta.-1,3-glucan.
[0219] In conducting the procedure described in this Example it was
noted that the .beta.-1,3-glucan raw material is a very fine powder
with a particle size of less than 5 micron in dry form. In liquid
form, it forms a fine suspension with a particle size less than 2
microns. Accordingly, to fully capture the active ingredients, a
more soluble hydrogel based gelatin has to be used.
[0220] It was further noted that in most cases a better hydrogel is
obtained using gelatin. However, gelatin is comparatively expensive
and pregel starch, for example pregel maize starch, may provide a
more economical base for said hydrogel.
[0221] A unit dose is prepared by mixing 750 mg of B&B
Preparation with 8.5 g of water to make a 10 ml paste.
[0222] The recommended dosage for prevention of diarrhoea in
piglets is 10 ml at day one of birth, repeat dosing in day five. In
farms with a serious history of diarrhoea, dosing may be repeated
at day ten and day thirteen.
Example 2d(i)
Field Trials Involving the Use of B&B Preparation
[0223] A field trial was conducted in Shangdong, China, to test the
efficacy of Bromelain Plus B glucan Preparation in prevention and
treatment of diarrhoea in piglets.
[0224] B&B Preparation was prepared as in Example 2d. Other
medications used in the trial were those standard in the field of
animal farming and management.
[0225] The trial was conducted at the Breeding Good Pig Farm of
Dezhou Husbandry Bureau, which produces over 10,000 pigs annually
and which has an incidence of diarrhoea of approximately 40% to
50%.
[0226] Nine litters randomly selected were divided into three
groups (three litters/group). Two groups were designated as
treatment groups, and the third group as a control. A total of
ninety seven piglets (Large White York piglets of mixed sex) were
included in the trial.
[0227] Litters were monitored for a period of twenty six days from
the time of first administration of B&B Preparation.
[0228] All piglets in treatment groups and control group were
vaccinated and given the same medication when sick (presenting
diarrhoea and/or associated symptoms).
[0229] The trial design is summarised in Table 7 below.
12TABLE 7 No of Given dose Groups pigs (5 ml) Appendix Treatment 1
34 Day 1 one dose, Monitor the efficacy repeat in day 5 of yellow
scours Treatment 2 31 Day 1 one dose, Monitor the efficacy repeat
in day 5, 10 of yellow and and 13. white scours Control 32 No
Bromelain Normal medications Preparation given
[0230] Results of the trial are summarised in Table 8 below.
13TABLE 8 MDWG (g) Number of Incidence (mean daily Mor- Groups
piglets of scour (%) weight gain) tality Comments Treatment 1 34
5.1 164 1 Scour Treatment 2 31 4.2 178 1 Starvation Control 32
17.59 169 1 Scour
[0231] As is seen from Table 8, the results of the trial
demonstrate that the incidence of scours in trial groups are 5.10%,
4.2% and 17.59% for treatment group 1, treatment group 2 and the
control group respectively. In other words, there was an observed
reduction in the incidence of diarrhoea of approximately 70%
between the treatment groups and the control groups. This
demonstrated B&B Preparation has a remarkable efficacy in the
prevention and/or treatment of diarrhoea in piglets.
[0232] No significant difference was observed in mean daily weight
gains (MDWG) between groups (164 g per day, 178 g per day, and 169
g per day).
[0233] Furthermore, the results suggest that administering the
preparation twice provides more efficient improvement of the
animal's health.
[0234] Overall, this trial indicates that B&B preparation
according to the invention has efficacy for preweaning scour, and
against non specific E. coli diarrhoea.
[0235] The inventors believe the formulation will have application
to other animals, including humans. Those of ordinary skill in the
art to which the invention relates will readily be able to modify
or adapt the formulation such that it is suitable for
administration to animals other than pigs.
Example 3
Slow Release Sublingual Stabilized Biological
Example 3a
Slow Release Sublingual Stabilized .beta.-1,3-Glucan Tablets
[0236] As previously discussed, .beta. glucans are effective
orally. However, when administered orally a substantial dosage is
generally required to achieve the desired immunomodulatory effect.
A dosage range of anywhere between 10 mg to 2000 mg per day has
been recommended, depending on the source of .beta. glucan. The
great range in recommended dosages it thought to be due to
variation in purity, and bioavailability, of the .beta. glucan
products on the market.
[0237] The present example provides a formulation of slow release
sublingual .beta.-1,3-glucan which when properly stabilized (via
the process of the present invention, for example) and delivered to
a specific mucosal surface, may be clinically active at a dose of
10 mg per day.
[0238] A product according to the present example may be suitable
for the treatment or alleviation of symptoms of an allergic
condition, for example, hayfever.
[0239] An example formula for a slow release sublingual
.beta.-1,3-glucan tablet, processed according to the invention, may
comprise the following constituents:
14 4 kg Batch Hydrogel core Gelatin 2.527 kg Polyvinylpyrrolidone
(Povidone) 0.400 kg Egg Albumin 0.400 kg Coating Liquid
.beta.-1,3-glucan 0.200 kg Mannitol 0.200 kg Propylene glycol 0.150
kg Gelatin (succinylated) 0.050 kg Standard sodium Phosphate buffer
to pH 7 0.073 kg Purified water to 2.000 kg
[0240] A slow release sublingual .beta.-1,3-glucan tablet of this
example was prepared according to the process of the invention as
follows:
[0241] 1 Huttlin Turbojet sterilised using heat (180.degree. C.) as
instructed by the equipment manufacturer.
[0242] 2 Hydrogel core material loaded into the Huttlin Turbojet
chamber by vacuum, fluidized and heated up to 60.degree. C. for one
hour.
[0243] 3 Hydrogel core temperature to reduced to 40.degree. C.
[0244] 4 Content of chamber fluidized at a rate of 300 cubic metres
per hour.
[0245] 5 .beta.-1,3-glucan coating turbojet coated onto the
hydrogel core under saturated moisture conditions at 25
g/minute.
[0246] 6 Resultant product dried to less than 3% moisture
content.
[0247] 7 Each 200 mg microcapsule contains 10 mg of
.beta.-1,3-glucan.
[0248] 8 Product compressed into 200 mg tablets according to
standard procedures used in the art.
[0249] 9 Product packed in nitrogen flushed aluminium/aluminium
foil pack and stored at a temperature not exceeding 25.degree.
C.
[0250] The tablets of this example have a slow dissolution rate
(less than minutes) due to the presence of a high percentage of
gelatin, polyvinylpyrrolidone (Povidone) and albumin in the
hydrogel core. This combination is ideal for slow release products
which allow the active material to have continuous contact with the
target absorption site, such as the oral mucosal membrane.
[0251] The recommended dosage regime for a product according to
this example, where it is used for the treatment or alleviation of
an allergic condition, is: one tablet dissolved under the tongue
daily, for four weeks prior to spring and continue for six months
thereafter. It is recommended that no food or drink be taken
fifteen minutes before or after medication.
[0252] A product according to this example was trialed by a 49 year
old female (Mrs Y) and a 39 year old male (Mr X) both of whom
suffered severe hay fever for many years. It was determined using
skin sensitivity tests that both subjects suffered from allergic
reactions to rye grass, pollen and house dust.
[0253] After taking the tablets of the present example in the dose
recommended above, both subjects reported that the incidence of
sneezing, itchy eyes and runny nose were minimal this spring
season, compared to previous years. Both subjects requested that
they be able to repeat the treatment in the following year to
determine whether their symptoms may be completely cured.
[0254] While not wishing to be bound to any particular theory, the
inventors of the present invention believe the sublingual use of
.beta.-1,3-glucan probably desensitised the immune system so that
the inflammatory response was down regulated.
[0255] The .beta.-1,3-glucan sublingual tablet of the present
example was also given to one severe asthmatic male aged 40 (Mr Z)
who has to use bronchodilator spray and corticosteroid medication
consistently. After two weeks of using the medication (one tablet
per day, dissolved under the tongue), the wheezing incidence was
greatly reduced and the frequency of the need to use the
bronchodilator spray and corticosteroid medication was halved.
However, while the medication was useful to reduce his asthmatic
conditions, for some unknown reasons, there were incidences of nose
bleeding. The .beta.-1,3-glucan medication was stopped
accordingly.
Example 4
Stabilization of Biologically Active Proteins
[0256] As previously mentioned herein biological proteins and
peptides have wide application in a number of industries, including
the pharmaceutical industry. However, many of these proteins may be
unstable at storage temperatures, such as room temperature.
[0257] The efficacy of the present invention in producing a stable
protein product is demonstrated in this example using Interferon
however, it will be appreciated that it is equally applicable to
the preparation of other proteins or peptides.
[0258] Interferon .alpha., .beta. and .beta. are antiviral,
antitumour and immunity modulating proteins. The method commonly
used to introduce exogenous interferon into the body of an animal
is by injection. Natural and recombinant interferon .alpha. 2a and
2b are commercially available as 3 million to 10 million IU
injections for treatment of viral and tumour diseases. All
commercial interferon injection products require storage at
approximately 4.degree. C. to 8.degree. C. because they are
unstable at elevated temperatures.
[0259] Administration of interferon by injection, at 3 million to 5
million IU dosages, are associated with significant side effects.
In addition, as interferon is not a native blood protein, it is
quite common that a patient may mount an immune response thereto,
after a few injections. Accordingly, subsequent dosages need to be
significantly increased in order for the interferon to have effect.
This in turn may worsen the side effects. Further, when
administered by injection, exogenous interferon will be carried via
the blood to the liver and quickly metabolised.
Example 4a
Stabilized Interferon .alpha. 3 Million IU Injection
[0260] The present example provides an interferon injection which
is stable at room temperature.
[0261] The stabilized interferon injection formulation comprises
the following components:
15 4 kg Batch Hydrogel core Gelatin 3.208 kg Polyvinylpyrrolidone
(Povidone) 0.400 kg Coating Liquid Interferon .alpha.2 40 billion
IU Mannitol 0.200 kg Propylene glycol 0.075 kg Gelatin
(succinylated) 0.025 kg Glycine 0.012 kg Egg Albumin 0.001 kg
Standard sodium phosphate buffers to pH 7 0.073 kg Water for
injection to 2.000 kg
[0262] It will be appreciated that the term "water for injection"
is one standard in the art. It refers to a standard grade of water
suitable for use in formulating injectable compositions, as
described in standard pharmacopoeia.
[0263] The stabilized interferon injection of the present example
was prepared according to the invention using the following
steps:
[0264] 1 Interferon .alpha., glycine, mannitol, gelatin
succinylated, propylene glycol, ascorbic acid and buffers are
dissolved in purified water then filtered through 0.22 micron
membrane filter. Albumin was added and made up to weight with water
for injection.
[0265] 2 Huttlin Turbojet chamber sterilised using heat
(180.degree. C.) as instructed by the equipment manufacturer.
[0266] 3 The Huttlin apparatus was switched to circulating filtered
nitrogen mode.
[0267] 4 Hydrogel core material loaded into the Huttlin Turbojet
chamber by vacuum, fluidized and heated up to 60.degree. C. for one
hour.
[0268] 5 Hydrogel core product temperature reduced to 40.degree.
C.
[0269] 6 Content of chamber fluidized at a rate of 300 cubic metres
per hour.
[0270] 7 Interferon .alpha. coating turbojet sprayed onto the
hydrogel core under saturated moisture conditions at a rate of 25
g/minute.
[0271] 8 Resultant product dried to less than 2% moisture
content.
[0272] 9 Product packaged in nitrogen flushed injection vials under
aseptic fill conditions.
[0273] Each 50 mg of the microcapsules prepared according to the
present example contains 3 million IU interferon .alpha..
Example 4b
Stabilized Interferon .alpha. 60.000 IU Pessaries
[0274] Room temperature stabilized Interferon pessaries can be made
to prevent and treat papilloma infections and other viral diseases
in the vaginal surface.
[0275] The stabilized interferon pessaries formulation of the
present example comprised the following components:
16 4 kg Batch Hydrogel core Polyvinylpyrrolidone (Povidone)/Acetic
acid ethenyl polymer 3.508 kg Coating Liquid Interferon .alpha.-2
40 million IU Mannitol 0.200 kg Propylene glycol 0.150 kg Gelatin
(succinylated) 0.050 kg Glycine 0.012 kg Egg Albumin 0.001 kg
Standard sodium phosphate buffers to pH 7 0.073 kg Purified water
to 2.000 kg
[0276] The stabilized interferon pessaries of the present example
were prepared according to the invention using the following
steps:
[0277] 1 Interferon .alpha., glycine, mannitol, gelatin
succinylated, propylene glycol, and buffers were dissolved in water
then filtered through 0.22 micron membrane filter. Albumin was
added and made up to weight with water for injection.
[0278] 2 Huttlin Turbojet heat sterilized at 180.degree. C. as
instructed by the equipment manufacture.
[0279] 3 Apparatus switched to circulating filtered nitrogen
mode.
[0280] 4 Hydrogel core material loaded into the Huttlin Turbojet
chamber by vacuum, fluidized and heated up to 60.degree. C. for one
hour.
[0281] 5 Hydrogel core product temperature reduced to 40.degree.
C.
[0282] 6 Contents of the chamber fluidized at a rate of 300 cubic
metres per hour.
[0283] 9 Interferon .alpha. coating turbojet sprayed onto the
hydrogel core under saturated moisture conditions at a rate of 25
g/minute.
[0284] 8 Resultant product dried to less than 2% moisture
content.
[0285] 9 Product compressed into 1 g pessaries, according to
standard procedures.
[0286] 10 Product packaged in nitrogen flushed aluminium/aluminium
foil packs and stored at a temperature not exceeding 25.degree.
C.
[0287] Each 1 g microcapsule prepared according to this example
contains 60,000 IU interferon .alpha..
Example 4c
Stabilized Interferon A 2000 IU & Muramidase Sublingual
Tablet
[0288] Interferon .alpha. at ultra low dose (less than 10,000
IU/dose/adult) has been demonstrated to be an effective medication
in treatment of viral and tumour diseases.
[0289] Subcutaneously administered interferon .alpha. is now
considered to be the standard therapy for the management of
hepatitis B and C and there are a number of commercially available
products therefor; for example, Wellferon TM Injection (Glaxo
Wellcome), Intron-ATM Injection (Schering Plough), Roferon ATM
Injection (Hoffmann-La Roche) and Anferon (Hualida Tianjin, China).
However, a number of disadvantages may be associated with the use
of such products including, for example: self-administration by
patients is painful and requires training; high-doses may be
associated with a number of side effects including vomiting,
nausea, dizziness, nasal discharge and other flu like symptoms; the
cost of treatment may be considered high, for example a normal
course of treatment for hepatitis B & C with Roferon A
Injection would cost around AU$8000 in Australia; and the products
are not stable at room temperature and must be stored under
refrigeration.
[0290] Studies conducted over approximately the past ten years have
indicated that low doses of interferon .alpha. may be administered
via the oromucosal route (including sublingual administration) with
efficacy.
[0291] The present example provides a dosage form which may be
administered via the oromucosal route (buccal or sublingual).
[0292] The sublingual slow release formulation of the present
invention contains both interferon .alpha. (2000 IU) and muramidase
hydrochloride (50 mg) and is useful in the treatment or
amelioration of chronic viral diseases; muramidase hydrochloride is
an antiviral agent with a history of extensive use in Asian
countries.
[0293] The stabilized interferon .alpha. and muramidase sublingual
tablet formulation of the present example comprised the following
components:
17 4 kg Batch Hydrogel core Gelatin 1.480 k Polyvinylpyrrolidone
(Povidone) 0.400 kg Muramidase Hydrochloride 1.000 kg Coating
Liquid Interferon .alpha. 2b 40 million IU Mannitol 0.200 kg
Propylene glycol 0.150 kg Gelatin (succinylated) 0.500 kg Glycine
0.120 kg Egg Albumin 0.020 kg Ascorbic Acid 0.057 kg Standard
sodium phosphate buffers to pH 7 0.073 kg Purified water to 4.000
kg
[0294] The stabilized interferon .alpha. and muramidase sublingual
tablets of the present example were prepared according to the
invention using the following steps:
[0295] 1 Interferon .alpha., glycine, mannitol, gelatin
succinylated, propylene glycol, ascorbic acid and buffers were
dissolved in water for injection then albumin was added and made up
to weight with water for injection.
[0296] 2 Huttlin Turbojet chamber sterilised by heating at
180.degree. C. as instructed by the equipment manufacturer.
[0297] 3 Apparatus switched to circulating filtered nitrogen
mode.
[0298] 4 Hydrogel core material loaded into the modified Huttlin
Turbojet chamber via vacuum, fluidized and heated up to 60.degree.
C. for one hour.
[0299] 5 Hydrogel core product temperature reduced to 40.degree.
C.
[0300] 6 Content of apparatus chamber fluidized at a rate of 300
cubic metres per hour.
[0301] 7 Interferon .alpha. coating turbojet sprayed onto the
hydrogel core under saturated moisture conditions at a rate of 25
g/minute.
[0302] 8 Resultant product dried to less than 2% moisture
content.
[0303] 9 Microcapsules compressed, according to standard procedures
used in the art, into 200 mg tablets and packed in nitrogen flushed
aluminium/aluminium foil packs and stored at a temperature not
exceeding 25.degree. C.
[0304] Each 200 mg tablet produced according to this example
contains 2,000 IU interferon .alpha. 2b and 50 mg of muramidase
hydrochloride, has a slow dissolution profile and requires not less
than ten minutes to dissolve in the mouth. It is designed as a slow
release sublingual product.
[0305] A product according to the present example is recommended to
be administered for the prevention and/or treatment of chronic
viral infections, according to the following preferable dosage
regime: one tablet every two days dissolved under the tongue over
six months to one year.
[0306] The stability of the actives (interferon .alpha. 2b and
muramidase) within the tablets of three different batches of the
product produced according to this example was studied at three
different temperatures. The results are collected in Table 9
below.
[0307] The potency of each active was assessed according to
standard procedures used in the art. Briefly, the following steps
were taken:
[0308] 1 The interferon and muramidase were extracted from the
existing solid phase into a stable and buffered liquid medium.
[0309] 2 The liquid extracted was subjected to a Cytopathic Effect
Assay (CPE) to determine the antiviral activity of interferon in
the tablet (according to the current method of British/European
Pharmacopoeia 2000).
[0310] 3 The liquid extract was subjected to HPLC analysis to
determine the quantity of muramidase in the tablets.
18 TABLE 9 IFN + Mu Bx 1 IFN + Mu Bx 2 IFN + Mu Bx 3 IFN + Mu Bx 1
IFN + Mu Bx 2 IFN + Mu Bx 3 IFN .alpha. 2b IFN .alpha. 2b IFN
.alpha. 2b Muramidase HCl Muramidase HCl Muramidase HCl IU IU IU mg
mg mg 4.degree. C. zero time 2200 1800 2200 47.5 50.0 47.5 3 months
2400 2200 2200 47.5 50.0 47.5 6 months 1400 3200 3000 48.0 48.5
46.0 9 months 2000 2400 1800 48.0 49.0 47.5 12 months 3000 1600
3550 46.5 49.0 49.0 24 months 2400 2200 2400 47.0 48.5 47.5
25.degree. C. zero time 2200 1800 2200 47.5 50.0 47.5 3 months 2800
2200 2400 47.5 50.0 47.5 6 months 1000 1400 2850 48.0 49.0 48.0 9
months 1600 2200 1800 48.0 49.5 48.5 12 months 2200 1850 2000 49.0
46.5 48.5 24 months 2400 2200 2200 48.0 47.5 48.0 35.degree. C.
zero time 2200 1800 2200 47.5 50.0 47.5 3 months 2400 2200 2000
47.5 50.0 47.5 6 months 2800 2000 2200 48.5 48.5 48.0 9 months 2000
1600 3800 48.0 48.5 47.5 12 months 2400 1800 3000 48.5 48.5 48.0 24
months 2000 2400 2000 48.0 48.5 48.0
Example 4
Stabilized Interferon .alpha. Nasal Spray for Prevention of Cold,
Flu and Other Respiratory Diseases
[0311] Interferon .alpha. is known to be effective against viral
respiratory diseases. Clinical studies in animals, including
humans, have demonstrated interferon .alpha. nasal spray at a dose
around a few hundred units to over one million units is effective
against respiratory infections, including those associated with the
flu and colds. However, at high dosages nose bleeding and flu-like
symptoms may be observed.
[0312] Correctly formulated interferon .alpha. 2b in aqueous phase
can be stable up to one month, but not more than two months, at
room temperature according to the information supplied by
manufacturers of interferon .alpha. 2b. Accordingly, interferon
nasal sprays for the treatment and/or prevention of colds, the flu
and other respiratory diseases, in a ready to use liquid form, with
a viable commercial shelf life (of approximately twelve to eighteen
months for example) are unavailable.
[0313] A viable alternative is to have the principal active
ingredient (interferon .alpha.) presented as a pre-constituted room
temperature stable tablet. Accordingly, just prior to use the
interferon .alpha. tablet may be added to a nasal spray bottle
containing an acceptable liquid diluent. The reconstituted solution
would have a shelf life of around four weeks at room temperature;
which shelf life would be suitable for the length of treatment of
most respiratory infections. The user may discard the bottle at the
completion of treatment.
[0314] Accordingly, the present example presents a consumer product
containing two components:
[0315] 1 A foiled packed interferon tablet containing 50,000 IU of
stabilized interferon .alpha.; prepared according to the process of
the invention; and
[0316] 2 A bottle containing 5 ml of an acceptable diluent, and
having a screw on nasal spray applicator.
[0317] It is preferred that the nasal spray applicator sprays a
metered dose of 0.1 ml of solution. According to the present
example, at this dosage rate, 1000 IU interferon .alpha. would be
administered with each spray.
[0318] A formulation according to the present example is preferably
administered at a rate of one 0.1 ml (1000 IU) spray per nostril
daily starting just prior to cold and flu season.
[0319] The stabilized tablets of the present example comprise the
following components:
19 4 kg Batch Hydrogel core Polyvinylpyrrolidone (Povidone)/Acetic
acid ethenyl polymer 3.508 kg Coating Liquid Interferon .alpha. 40
million IU Mannitol 0.200 kg Propylene glycol 0.150 kg Gelatin
(succinylated) 0.050 kg Glycine 0.012 kg Egg Albumin 0.001 kg
Standard sodium phosphate buffer to pH 7 0.073 kg Purified water to
2.000 kg
[0320] The stabilized tablets of the present example were prepared
according to the invention using the following steps:
[0321] 1 Interferon .alpha., glycine, mannitol, gelatin
succinylated, propylene glycol, and buffers are dissolves in water
for injection then filtered through 0.22 micron membrane filter.
Add albumin and make up to weight with water for injection.
[0322] 2 Huttlin Turbojet chamber sterilized by heat treatment at
180.degree. C. as instructed by the equipment manufacture.
[0323] 3 Apparatus switched to circulating filtered nitrogen
mode.
[0324] 4 Hydrogel material core loaded into the Huttlin Turbojet
chamber via vacuum, fluidized and heated up to 60.degree. C. for
one hour.
[0325] 5 Hydrogel core product temperature reduced to 40.degree.
C.
[0326] 6 Contents of the apparatus chamber fluidized at a rate of
300 cubic metres per hour.
[0327] 7 Interferon .alpha. coating turbojet sprayed onto the
hydrogel core under saturated moisture conditions at a rate of 25
g/minute.
[0328] 8 Resultant product dried to less than 2% moisture
content.
[0329] 9 Microcapsules compressed, according to standard procedures
in the art, into 200 mg tablets.
[0330] 10 Tablets packaged into nitrogen flushed
aluminium/aluminium foil packs and stored at a temperature not
exceeding 25.degree. C.
[0331] Each 200 mg tablets produced according to this example
contains 50,000 IU interferon .alpha..
[0332] The constituents of the liquid diluent formulation of the
present example are provided in Table 10 below.
20 TABLE 10 % w/w Gelatin BP/Eur.P 0.10 Povidone BP/Eur.P 0.10
Disodium edetate BP/Eur.P 0.14 Polysorbate 80 BP/Eur.P 0.20 Dextran
45,000 BP/Eur.P 0.22 Sodium dihydrogen phosphate BP/Eur.P
(Anhydrous Weight) 0.27 Disodium hydrogen phosphate BP/Eur.P
(Anhydrous Weight) 0.58 Glycine BP/Eur.P 0.10 Sodium propyl
hydroxybenzoate BP/Eur.P 0.03 Sodium methyl hydroxybenzoate
BP/Eur.P 0.09 Albumin BPC 0.05 TOTAL 2.93 Purified water BP/Eur.P
to 100%
Example 4e
Stabilized Interferon .alpha. Nasal Spray for Treatment of Cold,
Flu and Other Respiratory Diseases
[0333] The present example, provides an alternative diluent
formulation containing additional active ingredients that may help
relieve cold and flu symptoms.
[0334] The consumer product of the present example contains the two
components listed in Example 4d; the tablet containing the
interferon .alpha. and the diluent within a spray applicator bottle
or the like. Similarly, the reconstituted interferon nasal spray
will contain 1000 IU interferon .alpha./0.1 ml.
[0335] The preferred dosage regime of the present example is: one
0.1 ml (1000 IU) spray per nostril morning and night when mild flu
or cold symptoms appears. Continue treatment for five to ten
days.
[0336] The tablet component of the present example is prepared
according to process of Example 4d.
[0337] The diluent formula of the present formulation is given in
Table 11 below:
21 TABLE 11 % w/w Oxymetazoline hydrochloride USP 0.05
Dexchlorpheniramine meleate USP 1.00 Gelatin BP/Eur.P 0.10 Povidone
BP/Eur.P 0.10 Disodium edetate BP/Eur.P 0.14 Polysorbate 80
BP/Eur.P 0.20 Dextran 45,000 BP/Eur.P 0.22 Sodium dihydrogen
phosphate BP/Eur.P (Anhydrous Weight) 0.27 Disodium hydrogen
phosphate BP/Eur.P (Anhydrous Weight) 0.58 Glycine BP/Eur.P 0.10
Sodium propyl hydroxybenzoate BP/Eur.P 0.03 Sodium methyl
hydroxybenzoate BP/Eur.P 0.09 Albumin BPC 0.05 TOTAL 2.93 Purified
water BP/Eur.P to 100%
Example 4f
Stabilized Interferon .alpha. Eye Wash or Drops for Prevention of
Red Eve Disease
[0338] As previously discussed, correctly formulated aqueous
interferon .alpha. may be stable up to one month, but not two, at
room temperature. Accordingly, interferon eye washes or drops, in a
ready to use liquid form, with a viable commercial shelf life (of
approximately twelve to eighteen months, for example) are
unavailable.
[0339] A viable alternative is to have the principal active
ingredient (interferon .alpha.) presented as a preconstituted room
temperature stable tablet. Accordingly, just prior to use the
interferon .alpha. tablet may be added to an eye wash, or eye drop,
bottle containing an acceptable liquid diluent. The reconstituted
solution would have a shelf life of around four weeks at room
temperature. The user may discard the bottle at the completion of
treatment.
[0340] The consumer product of the present example contains the two
components listed in Example 4d; the tablet containing the
interferon .alpha. and the diluent (albeit in an appropriate eye
wash or drop bottle, in this example). Similarly, the reconstituted
interferon eye wash or drops contain 1000 IU interferon .alpha./0.1
ml, with 0.1 ml being the preferred single administration dose.
[0341] The preferred dosage regime of this example is: one 0.1 ml
(1000 IU) spray/drop per eye, twice daily for treatment of red
eye.
[0342] The interferon .alpha. tablet formulae of the present
example is formulated and processed according to that described in
Example 4d above.
[0343] The diluent formulation of the present example is provided
in Table 12 below. It will be appreciated that this liquid diluent
is preferably autoclaved, or otherwise sterilised.
22 TABLE 12 % w/w Povidone BP/Eur.P 0.10 Disodium edetate BP/Eur.P
0.14 Polysorbate 80 BP/Eur.P 0.20 Dextran 45,000 BP/Eur.P 0.22
Sodium dihydrogen phosphate BP/Eur.P (Anhydrous Weight) 0.27
Disodium hydrogen phosphate BP/Eur.P (Anhydrous Weight) 0.58
Glycine BP/Eur.P 0.10 Sodium propyl hydroxybenzoate BP/Eur.P 0.03
Sodium methyl hydroxybenzoate BP/Eur.P 0.09 Albumin BPC 0.05 TOTAL
2.93 Purified water BP/Eur.P to 100%
Example 4g
Stabilized Interferon Skin Spray--Same Formulation as Eyewash
[0344] A skin spray, for wound healing, for example, was formulated
according to Example 4f. However, in this example, the liquid
diluent was presented in an appropriate container for delivery to
the skin.
Example 4h
Stabilized Erythropoietin (EPO) for Sublingual Delivery
[0345] Stabilized formulations of EPO were prepared in tablet form
according to the following process.
23 % w/w Hydrogel Core Dextrose (anhydrous) BP 46.52 Starch BP
(anhydrous wt) 20.00 Gelatin BP/Eur.P(anhydrous wt) 20.00
Carmellose BP (anhydrous wt) 2.00 Coating Liquid EPO (Epoetin Alfa)
250,00 IU Dextran 40,000 BP/Eur.P 0.600 Sodium Dihydrogen Phosphate
BP/Eur.P 0.042 Disodium Hydrogen Phosphate BP/Eur.P (anhydrous wt)
0.057 Glycine BP/Eur.P 0.030 Trehalose 0.600 Sodium Edetate BP
0.025 Propylene Glycol BP 0.050 Albumin BPC 5.030 Sodium Chloride
BP 0.046 Leucine USP 3.000 TOTAL 100% Purified Water BP/Eur.P
to
[0346] The sublingual EPO tablets of the present example were
prepared according to the invention using the following steps:
[0347] 1 EPO, dextran, glycine, trehalose, sodium edetate,
propylene glycol, sodium chloride, leucine and buffers were
dissolved in water for injection then albumin was added and made up
to weight with water for injection.
[0348] 2 Huttlin Turbojet heat sterilised at 180.degree. C. as
instructed by the equipment manufacturer.
[0349] 3 Apparatus switched to circulating filtered nitrogen
mode.
[0350] 4 Hydrogel core material loaded into the modified Huttlin
Turbojet chamber via vacuum, fluidized and heated up to 60.degree.
C. for one hour.
[0351] 5 Hydrogel core product temperature reduced to 40.degree.
C.
[0352] 6 Contents of the chamber fluidized at a rate of 300 cubic
metres per hour.
[0353] 7 EPO coating turbojet sprayed onto the hydrogel core under
saturated moisture conditions at a rate of 25 g/minute.
[0354] 8 Resultant product dried to less than 2% moisture
content.
[0355] 9 Product compressed into 200 mg tablets according to
standard procedures.
[0356] An animal study was conducted using the formulations so
produced. Rats were divided into four groups. Group 1 received 50
IU EPO via subcutaneous injection on day one, and blood samples
were taken for analysis on days two, four and six for reticulocyte
determination. This mode of administration mimics the current
delivery route for EPO therapy.
[0357] In Group 2, rats received a daily dose of 125 IU EPO via
sublingual delivery with the formulation of this example on days
one, two and three. Blood samples were taken on days two, four, six
and eight for reticulocyte determination.
[0358] The Group 3 rats received a daily dose of 125 IU EPO via
sublingual delivery on days one, two, three, four and five. Blood
samples were taken on days two, four, six, eight and ten for
reticulocyte determination.
[0359] Controlled rats received either a subcutaneous injection of
saline of the same volume as the 50 IU EPO subcutaneous injection
for Group 1. Control Group 2 received a formulation according to
this example containing no EPO.
[0360] Results
[0361] The results are shown graphically in FIG. 1, with control
animals being represented in the "normal range" figures
presentation. Rats received sublingual EPO showed demonstrably
elevated reticulocyte counts above the normal range for control
animals, indicating that the EPO delivered sublingually was
active.
[0362] The subcutaneously injected EPO showed peak reticulocyte
counts after two days, which rapidly fell off to the normal range
at four days.
[0363] The formulation of this example was tested for stability,
and results are shown in FIG. 2. As shown in this figure, the EPO
containing sublingual tablet is stable at room temperature for at
least nine months. The activity of EPO was determined by human EPO
immunoassay and found to be fully biologically active, as measured
by reticulocyte counts in rats.
[0364] This example shows that stabilized proteins, such as
erythropoietin, are released and absorbed through contact with
mucosal surface of the body, including sublingual delivery, nasal
delivery and vaginal delivery. These mucosal areas are rich in
lymphoidal tissue which allows transport of the proteins into the
body for treatment of anemia caused by, for example, kidney
failure, AIDS, cancer, genetic diseases, operation and
menstruation.
[0365] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features, and
compositions referred to or indicated in this specification,
individually or collectively, and any and all combinations of any
two or more of said steps or features.
[0366] Particularly, it will be appreciated by those of general
skill in the art to which the present invention relates, that while
the present invention has been described and exemplified with
reference to the preparation of specific proteins and
micro-organisms it is equally applicable to the preparation of any
cells and/or proteins or peptides of interest. For example, the
process of the invention may be readily applicable to the
preparation of hormones, cytokines, and growth factors such as
human or animal growth hormone, or derivatives thereof;
erythropoietin (EPO) including those produced by recombinant
techniques for example Epoetin .alpha., Epoetin .beta., and Epoetin
.gamma.; calcitonin; interferons including .alpha.- and .beta.- and
.gamma.-interferons; interleukins such as IL 2; insulins; colony
stimulating factors such as G-CSF and GM-CSF. Examples of enzymes
which may be used in the invention include streptokinase,
muramidase, pancreas, amylase, protease, lypase, cellulase,
bromelain, papain and the like. The formulations may include two or
more different enzymes.
[0367] The specification provides examples of preferred dosage
rates for the use of a number of the novel formulations made
according to the invention. Alternative dosages and concentrations
of active therein are envisaged by the inventors and those of
general skill in the art to which the invention relates will
readily be able to formulate products, according to the present
invention, which have alternative concentrations of active
therein.
[0368] Biological proteins being stabilized as microcapsules in
this invention are also able to be released when in contact with
mucosal surfaces of the body eg sublingual, nasal, vaginal. These
mucosal areas are rich in lymphoidal tissue which allow transport
of the protein into the body. Investigations of various proteins as
subcutaneous injections by Charman et al (J. Pharm. Sci., Vol 89,
pages 168-177 (2000)) and later by McLennan (APSA 2001 Conference
Proceedings), showed lymphatic absorption of proteins increased
with molecular weight of the protein and the lymphatic system was
significant in contributing to the overall systemic availability of
proteins administered via subcutaneous injections. The stabilised
protein microcapsules can allow more patient acceptable dosage
forms, such as a sublingual tablet, nasal spray or vaginal pessary,
than subcutaneous injection.
[0369] Further, it will be appreciated that a product according to
the invention may be manipulated or further formulated in order to
arrive at a desired dose form. For example, the microcapsules of
the invention may be encapsulated to form standard capsule unit
doses, or may be combined with various standard excipients and
diluents used in the art, to form tablets or liquid formulations,
for example. Those of skill in the art will appreciate many other
ways in which the micro-capsules of the invention may be further
formulated and that they are contemplated by the inventors of the
present invention.
[0370] Finally, it is contemplated by the inventors of the present
invention that the novel process described herein may be used to
prepare other materials and to manipulate materials to particular
ends. In particular, it is envisaged that the inventive process may
be manipulated to allow for enteric coating of microcapsules using
solvents or aqueous methods such as sodium salts of cellulose
acetate phthalate, to create sustained release properties by
changing the core material polymers to a higher molecular weight or
by using a combination of hydrogel core such as high molecular
weight gelatin, polyvinyl pyrrolidone, alginates, carboxymethyl
cellulose, various cellulose derivative, polyethylene glycols,
albumin, karregreenin (one of ordinary skill in the art of
formulation will be able to provide various combinations to create
a desired release profile), create time release properties by
varying the nature of polymers used and the thickness of the
coatings and to allow for micro-distribution of trace materials
among large amount of solid mass.
[0371] Titles, headings, or the like are provided to enhance the
reader's comprehension of this document, and should not be read as
limiting the scope of the present invention.
[0372] The entire disclosures of all applications, patents and
publications, cited above and below, if any, are hereby
incorporated by reference.
[0373] The reference to any prior art in this specification is not,
and should not be taken as, an acknowledgment or any form of
suggestion that that prior art forms part of the common general
knowledge in the field of endeavour.
[0374] Throughout this specification, unless the context requires
otherwise, the word "comprise", and variations such as "comprises"
and "comprising", will be understood to imply the inclusion of a
stated integer or step or group of integers or steps but not the
exclusion of any other integer or step or group of integers or
steps.
* * * * *