U.S. patent application number 13/528215 was filed with the patent office on 2012-10-11 for encapsulated bacteriophage formulation.
Invention is credited to Rainer Engelhardt, Kishore Murthy.
Application Number | 20120258175 13/528215 |
Document ID | / |
Family ID | 36318849 |
Filed Date | 2012-10-11 |
United States Patent
Application |
20120258175 |
Kind Code |
A1 |
Murthy; Kishore ; et
al. |
October 11, 2012 |
ENCAPSULATED BACTERIOPHAGE FORMULATION
Abstract
An encapsulated bacteriophage formulation and a method for
preparing encapsulated bacteriophage formulation is provided. The
method for producing the encapsulated bacteriophage composition
involves injection of a molten coating substance comprising stearic
acid and palmitic acid present at a ratio of 50:50, into a
granulator chamber containing immobilized bacteriophages. The
immobilized bacteriophage are agitated by rotation of a base of the
chamber and swept by a flow of air at a temperature of between
10.degree. C. and 50.degree. C.
Inventors: |
Murthy; Kishore; (Ottawa,
CA) ; Engelhardt; Rainer; (Ottawa, CA) |
Family ID: |
36318849 |
Appl. No.: |
13/528215 |
Filed: |
June 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11666706 |
May 20, 2008 |
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PCT/CA2005/001679 |
Nov 2, 2005 |
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13528215 |
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60624576 |
Nov 2, 2004 |
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Current U.S.
Class: |
424/490 ;
424/93.6; 427/2.1 |
Current CPC
Class: |
A23K 50/80 20160501;
A61P 17/02 20180101; A23K 50/10 20160501; A61K 35/76 20130101; A61P
31/04 20180101; A23K 50/75 20160501; Y02A 40/818 20180101; A23K
20/195 20160501; C12N 2795/00032 20130101; C12N 2795/00051
20130101 |
Class at
Publication: |
424/490 ;
424/93.6; 427/2.1 |
International
Class: |
A61K 35/74 20060101
A61K035/74; B05D 7/00 20060101 B05D007/00; A61K 9/48 20060101
A61K009/48 |
Claims
1. A method for producing an encapsulated bacteriophage formulation
comprising, injecting a molten coating substance comprising stearic
acid and palmitic acid at a ratio of 50:50 at a temperature of
between about 20.degree. C. and about 120.degree. C. into a chamber
containing bacteriophage immobilized on skim milk powder, the
bacteriophage immobilized on skim milk powder agitated by rotation
of a base of the chamber and swept by a flow of air at a
temperature of between 10.degree. C. and 50.degree. C., the flow of
air having a speed such that the chamber temperature is between
about 30.degree. C. to about 55.degree. C., to produce an
encapsulated bacteriophage formulation.
2. The method according to claim 1, wherein the speed of rotation
of the base of the chamber is between 50 and 500 rpm.
3. The method according to claim 1, wherein the coating substance
has a melting point of between 30.degree. C. and 100.degree. C.
4. The method according to claim 1, wherein the injection
temperature is between 60.degree. C. and 120.degree. C.
5. The method according to claim 1, wherein the chamber temperature
does not exceed by more than 5.degree. C. a viability temperature
of the immobilized bacteriophages.
6. An encapsulated bacteriophage formulation comprising
bacteriophage immobilized on skim milk powder and encapsulated with
a coating substance comprising stearic acid and palmitic acid
present at a ratio of 50:50.
Description
[0001] This application is a Continuation application of U.S.
patent application Ser. No. 11/666,706, filed May 20, 2008, the
entire contents of which are hereby incorporated herein by
reference. U.S. patent application Ser. No. 11/666,706 is a
National Stage application of International Application No.
PCT/CA2005/001679, filed Nov. 2, 2005, the entire contents of which
are hereby incorporated herein by reference. International
Application No. PCT/CA2005/001679 claims the benefit of U.S.
Provisional Application No. 60/624,576, filed Nov. 2, 2004, the
entire contents of which are hereby incorporated herein by
reference.
[0002] The present invention relates to an encapsulated
bacteriophage formulation. More particularly, the present invention
pertains to an encapsulated bacteriophage formulation and methods
for preparing the encapsulated bacteriophage formulation.
BACKGROUND OF THE INVENTION
[0003] Bacteriophage therapy has the potential to provide an
effective method to control the undesirable multiplication of
various strains of bacteria. However, to be commercially viable,
the bacteriophages themselves must show a certain degree of
stability to allow for storage.
[0004] Various methods have been used to store and protect phage,
including freezing at low temperatures, lyophilising, and storing
in liquid medium. All methods have shown varying degrees of success
at maintaining a high titer of viable bacteriophages.
[0005] Prouty (1953, Appl Microbiol, 1:250-351) reported that
desiccated bacteriophage of lactic acid Streptococci remained
viable at 0.degree. C. for 42 months, at 37.degree. C. for 72
months and at 12.degree. C. and 25.degree. C. for at least 78
months. However, there is no mention of the effect of storing
desiccated bacteriophage on the titer of the bacteriophage.
[0006] Keogh and Pettingill (1966, Appl Microbiol, 14:4421-424)
show that bacteriophages for lactic acid Streptococci in the
presence of whey protein are resistant to freezing and cold
storage. Phage stored at 4.degree. C. and -18.degree. C. showed
little reduction in the bacteriophage titer; freeze-thaw cycles
also showed no significant loss of titer. Warren and Hatch (1969,
Appl Microbiol, 17:256-261) report a significant decrease in the
titer and viability of a bacteriophage suspension stored (without
stabilizers) at 4.degree. C., while storage at -20.degree. C. and
20.degree. C. resulted in the greatest survival of phage. They also
report that long term storage of bacteriophages at -20.degree. C.
tends to result in the formation of clumps.
[0007] Jepson and March (2004, Vaccine, 22:2413-2419) disclose that
a liquid suspension of bacteriophages (in either SM buffer or a
1/200 dilution of SM buffer in water) was stable for 6 months at
4.degree. C. and -70.degree. C., with the phage remaining
unaffected by freeze-thawing. Increased temperature, between
20.degree. C. and 42.degree. C., resulted in a significant loss of
titre. Lyophilisation and immediate reconstitution of
bacteriophages in the presence or absence of stabilizers resulted
in a loss of titre of 80-95%. Of the bacteriophages remaining
following lyophilization in the presence of dry skim milk powder,
storage at temperatures between 20.degree. C. and 42.degree. C.
resulted in a loss of titre similar to that of the liquid
suspension. However, lyophilization in the presence of trehalose
resulted in an increase in half-life of bacteriophage between
20.degree. C. and 42.degree. C. The effect of pH of the storage
medium was also examined. There was no change in bacteriophage
titer over a 24 hour period at pH 3-11. However, the titer dropped
rapidly when stored for 5 minutes at pH values below 2.4.
[0008] Freezing or lyophilisation of bacteriophage suspensions, or
bacteriophage suspensions optionally containing stabilizers, are
inconvenient methods that require specialized equipment and add to
the cost of a commercial preparation. While it may be in certain
circumstances desirable to be able to store bacteriophages in a
desiccated state, the process of lyophilization results in a
significant loss of titre. Alternative methods for bacteriophage
stabilization are required
SUMMARY OF THE INVENTION
[0009] The present invention relates to an encapsulated
bacteriophage formulation. More particularly, the present invention
pertains to an encapsulated bacteriophage formulation and methods
for preparing the encapsulated bacteriophage formulation.
[0010] It is an object of the present invention to provide an
encapsulated bacteriophage formulation.
[0011] The present invention provides a method for producing an
encapsulated bacteriophage formulation comprising,
[0012] injecting a molten coating substance comprising a 50:50
solution of palmitic and stearic acids, at a temperature of between
about 20.degree. C. and about 80.degree. C. into a chamber
containing bacteriophage immobilized on skim milk powder, the
bacteriophage immobilized on skim milk powder agitated by rotation
of a base of the chamber and swept by a flow of air at a
temperature of between 10.degree. C. and 50.degree. C., the flow of
air having a speed such that the chamber temperature is between
about 30.degree. C. to about 55.degree. C., to produce an
encapsulated bacteriophage formulation.
[0013] The present invention also pertains to the method described
above wherein the speed of rotation of the base of said chamber may
be between 50 and 500 rpm. In the method as described above, the
coating substance may also have a melting point of between
30.degree. C. and 80.degree. C. The present invention also pertains
to the above method, wherein the injection temperature may be
between 60.degree. C. and 120.degree. C. In the above method, the
temperature in the chamber may be between 30.degree. C. and
55.degree. C. Additionally, in the method as described above, the
proportion of the injected coating substance may be between 10 and
99% by weight of the encapsulated bacteriophage formulation.
[0014] The present invention also provides an encapsulated
bacteriophage formulation comprising an bacteriophage immobilized
on skim milk powder and encapsulated with a coating consisting of
stearic acid and palmitic acid present at a ratio of 50:50.
[0015] The encapsulated bacteriophage formulation of the present
invention is resistant to extended periods of exposure to low pH
that would otherwise render the bacteriophage non-viable.
Encapsulated antibacterial compositions of the present invention
may be used for any suitable purpose that requires a bacteriophage
preparation, for example, the encapsulated bacteriophage
composition may be added to animal feed and fed to an animal. In
this case, the encapsulation of the bacteriophages results in
protecting the bacteriophages from stomach acids and increasing the
duration of bacteriophage release within the gut of the animal. The
antibacterial compositions of the present invention may be used for
veterinary purposes.
[0016] This summary of the invention does not necessarily describe
all necessary features of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] These and other features of the invention will become more
apparent from the following description in which reference is made
to the appended drawings wherein:
[0018] FIG. 1 shows the effect of encapsulation on bacteriophage
activity. Phage titers before and after encapsulation are
shown.
[0019] FIG. 2A shows the effect of low pH on the stability of
encapsulated phages. Encapsulated phage titers were determined
before and after grinding. All phage concentrations have been
corrected for the weight of encapsulated material. FIG. 2B shows
the effect of low pH on the infectivity of phage. The phages were
neither immobilized nor encapsulated.
DESCRIPTION OF PREFERRED EMBODIMENT
[0020] The present invention relates to stabilized bacteriophage
formulations. More particularly, the present invention pertains to
stabilized bacteriophage formulations and methods for preparing
stabilized bacteriophage formulations.
[0021] The following description is of a preferred embodiment.
[0022] The present invention provides an encapsulated bacteriophage
formulation. The present invention also provides a method for
producing an encapsulated bacteriophage formulation comprising
encapsulating immobilized bacteriophages.
[0023] The encapsulated bacteriophage formulation may be used in a
variety of ways for the control of bacterial growth, and may be
used for a variety of applications. For example, the antibacterial
composition may be encapsulated and used as a feed additive or as
an oral treatment for the control of bacteria within a human, a
mammal, or an avian species.
[0024] The term "bacteriophage" or "phage" is well known in the art
and generally indicates a virus-like particle that infects
bacteria. Phages are parasites that multiply inside bacterial cells
by using some or all of the hosts' biosynthetic machinery, and can
either be lytic or non-lytic. The bacteriophages used in accordance
with the present invention may be any bacteriophage, lytic or
non-lytic, that is effective against a target bacterium of
interest. The target bacteria may be any type of bacteria, for
example but not limited to species and serotypes of, E. coli,
Streptococci, Humicola, Salmonella, Campylobacter, Listeria,
Staphylococcus, Pasteurella, Mycobacterium, Hemophilius,
Helicobacter, Mycobacterium, Mycoplasmi, Nesseria, Klebsiella,
Enterobacter, Proteus, Bactercides, Pseudomas, Borrelius,
Citrobacter, Propionobacter, Treponema, Shigella, Enterococcus,
Leptospirex, Bacillii including Bacillus anthracis and other
bacteria pathogenic to humans, animal, fish, birds, plants. If
desired, one of or a mixture or cocktail of identified differing
bacteriophages may be used against a single bacterial target, or
multiple bacterial targets.
[0025] The bacteriophage may be provided in a powder form as an
immobilized bacteriophage preparation. For example, the
bacteriophage may be adsorbed onto skim milk powder. Immobilized
bacteriophage may be obtained from GangaGen Life Sciences Inc.
(Ottawa, Canada).
[0026] By the term "encapsulated", it is meant that the immobilized
phages are coated with a substance that increases the phages'
resistance to the physico-chemical stresses of its biotic or
abiotic environment. The immobilized phages may be coated with a
mixture of palmitic and stearic acids, present at a ration of
50:50, in accordance with the method described in US publication
2003/0109025 (Durand et al., which is incorporated herein by
reference in its entirety). In this method, micro-drops of the
coating substance are injected into a chamber containing the
immobilized bacteriophages and rapidly cooled.
[0027] The coating layer exhibits a melting point between about
20.degree. C. and about 120.degree. C., for example between about
30.degree. C. and about 80.degree. C., or any temperature
therebetween. If the coating substance is to be ingested or used
for an oral application, then it is preferred that the substance is
a food grade substance. An example of a coating substance comprises
a mixture of stearic acid and palmitic acid present at a ratio of
50:50, for example, Stearine 50/50.TM. (obtained from Exaflor, Gif
sur Yvette, France). Other additive molecules may be added to the
coating substance including antioxidants, sugars, or proteins.
[0028] In the encapsulated bacteriophage formulation of the present
invention, the proportion of coating material in relation to the
quantity of immobilized bacteriophages is between about 10% and
about 99% by weight, or any amount therebetween, for example
between about 30% and about 80%. For example, the proportion of
coating material in relation to the quantity of immobilized
bacteriophages may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95 or 99% by weight, or any amount in a
range defined by any two percentages just recited. This parameter
may be adjusted according to the intended final application,
depending on the desired rate of release of the immobilized
bacteriophage composition.
[0029] The coating method uses a granulator (GLATT..TM. granulator
model CRG200). As would be known to a person of skill in the art,
this device comprises a stainless steel chamber, the base of which
is made up of a rotatable disc that is powered by a motor. An
airflow is injected via the space between the base and the body of
the chamber. The air escapes from the chamber through a filter
placed in the upper portion of the chamber. The mass of immobilized
bacteriophages is agitated by the rotation of the disc. A nozzle
permits injection, by means of a pump, of the coating product kept
at a temperature above its melting point in a
temperature-controlled receptacle.
[0030] The coating substance is used in the granulator in a molten
form. The temperature of the molten coating substance placed in the
temperature-controlled receptacle can be about 30.degree. C. and
about 120.degree. C., for example between about 60.degree. C. and
about 120.degree. C., or any temperature therebetween; for example,
the temperature of the coating substance may be 30, 35, 40, 45, 50,
55, 60, 65, 70 75, 80, 85, 90, 95, 100, 105, 110, 115, or
120.degree. C. or any temperature in a range defined by any two
temperatures just recited. In any case, the temperature of the
coating substance ought to be greater than the melting point of
said coating substance, whether this is a pure product or a
mixture. For example, the temperature of the coating substance may
be at least about 5.degree. C., or at least about 10.degree. C.
above the meting temperature of the coating substance.
[0031] The temperature of the air sweeping the granulation chamber
is between about 10.degree. C. and about 50.degree. C., or any
temperature therebetween; for example, the temperature of the, air
may be about 10, 15, 20, 25, 30, 35, 40, 45, or 50.degree. C. or
any temperature in a range defined by any two temperatures just
recited. The temperature of the air sweeping the granulation
chamber is strictly controlled, so that at the moment of injecting
the molten coating, the rise in temperature experienced by the
immobilized bacteriophages does not exceed a few degrees, at
maximum about 5.degree. C.
[0032] The speed of rotation of the base plate and the rate of
injection of the coating are interdependent parameters, connected
to the mass of the products used. The speed of rotation is
generally between about 50 and about 500 rpm (rotation per minute),
or any speed therebetween; for example, the speed of rotation may
be about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,
350, 375, 400, 425, 450, 475, or 500 rpm, or any speed in a range
therebetween. The injection of the coating can be carried out with
the aid of one or more nozzles distributed over the periphery of
the chamber.
[0033] The encapsulated bacteriophage formulation of the present
invention exhibits desirable storage properties and may be used in
a variety of applications. For example, which is not to be
considered limiting in any manner, the antibacterial compositions
may be used for human, veterinary, aquacultural, and agricultural
applications. For example, the encapsulated bacteriophage
formulation may be mixed with the feed of livestock, birds,
poultry, domestic animals and fish, to aid in reducing the shedding
of target bacteria.
[0034] The present invention will be further illustrated in the
following examples.
EXAMPLES
Example 1
Encapsulation of Bacteriophage Compositions
[0035] Immobilized bacteriophages using skim milk as the matrix,
were obtained from GangaGen Life Sciences Inc (Ottawa, Canada), and
encapsulated generally as described in US publication 2003/0109025
(which is incorporated herein by reference) using a GLATT.TM.
granulator, model CRG200, with some modifications to preserve the
activity of the phages. Briefly, 400 g of immobilized phages and
1.2 kg of vegetable fatty acids comprising palmitic and stearic
acids and available as Stearine 50/50.TM. (obtained from Exaflor,
Gif sur Yvette, France) were used for encapsulation. The maximum
temperature attained by the encapsulated phage preparation was
39.degree. C.
[0036] Once the coating operation was complete, the encapsulated
particles were collected and stored in airtight containers. The
average particle size was between 100 and 1000 .mu.m.
[0037] The effect of encapsulation on the titer of bacteriophages
was determined by determining the activity of the immobilized
phages before ("Before", FIG. 1) and after ("After", FIG. 1)
encapsulation. For this analysis, encapsulated bacteriophages were
re-suspended, and ground using a blender. The re-suspended
encapsulated bacteriophages were blended in order to disrupt the
encapsulated particles and release the bacteriophages. 0.5 g of
encapsulated immobilized phage was mixed with 45.5 ml of
re-suspension media (LB Broth or RO Water), and 250 .mu.l of
antifoam agent was added to prevent foaming upon grinding. The
results of this analysis are shown in FIG. 1.
[0038] These results demonstrate that bacteriophages can be
recovered from an encapsulated bacteriophage composition, and
encapsulation does not inactivate the immobilized phage.
Example 2
Stability And Release of Encapsulated Bacteriophages
[0039] Phages were encapsulated as described in Example 1. The
release of phages upon physical or chemical disruption was tested
in the following manner: 0.5 g of encapsulated immobilized phage
was mixed with 45.5 ml of re-suspension media (LB Broth or RO
Water). 250 .mu.l of antifoam agent was used to prevent foaming
upon grinding. A control sample of encapsulated phages was prepared
as described above, but not subjected to grinding, to determine the
non-specific leaching of encapsulated bacteriophages within the
re-suspension medium.
[0040] The stability of the encapsulated bacteriophages at low pH
was also examined. After re-suspension (as outlined above), the
encapsulated phages were incubated for 30 or 60 min at pH 2.15,
neutralized to pH 7.0 using NaOH, then ground using a blender;
another sample (control) was resuspended and immediately ground.
Both the control and test samples were filter sterilized using a
0.45 .mu.m syringe filter prior to use.
[0041] FIG. 2A shows the results of these analyses. The data show
that resuspension of the encapsulated immobilized phage results in
phage concentrations of about 1E+08 pfu/g. Similarly, incubation of
the phages at pH 2.15 alone does not cause significant release of
phages (phage concentration of about 3E+07 pfu/g after 30 minutes,
or a phage concentration of about 4E+07 pfu/g after 60 minutes).
However, following grinding and disruption of the encapsulated
bacteriophage particles, the amount of phage released is about the
same amount as was loaded onto the milk powder for immobilization
(about 2E+09 pfu/g). Incubation of non encapsulated and non
immobilized phages at pH 2.15 for 30 and 60 minutes however
resulted in essentially complete loss of phage infectivity (FIG.
2B).
[0042] These results demonstrate that bacteriophages may be
released following disruption of encapsulated bacteriophage
particles. Furthermore, these results shows that encapsulated
bacteriophage may be exposed to a pH of 2.15 for prolonged period
of time, with little or no loss in activity (titer). The results
for non-encapsulated and non-immobilized bacteriophages are
consistent with the results of Jepson and March (2004, Vaccine,
22:2413-2419), where a dramatic loss of viability of bacteriophages
was observed after only 5 minutes at pH below pH 2.2. This loss in
activity is obviated by encapsulation of the bacteriophages as
described in the present invention.
[0043] All citations are hereby incorporated by reference.
[0044] The present invention has been described with regard to one
or more embodiments. However, it will be apparent to persons
skilled in the art that a number of variations and modifications
can be made without departing from the scope of the invention as
defined in the claims.
* * * * *