U.S. patent application number 14/412420 was filed with the patent office on 2015-07-02 for hydrogel vaccine formulations.
This patent application is currently assigned to Sigmoid Pharma Ltd.. The applicant listed for this patent is Sigmoid Pharma Limited. Invention is credited to Vincenzo Aversa, Ivan Coulter, Bernard Francis McDonald, Monica Torres Rosa.
Application Number | 20150182456 14/412420 |
Document ID | / |
Family ID | 46766220 |
Filed Date | 2015-07-02 |
United States Patent
Application |
20150182456 |
Kind Code |
A1 |
Coulter; Ivan ; et
al. |
July 2, 2015 |
HYDROGEL VACCINE FORMULATIONS
Abstract
This invention relates to compositions for delivering one or
more active ingredients, and more particularly to compositions,
e.g. beads, comprising a matrix material which matrix material
comprises a microorganism. In particular, the invention relates to
compositions comprising a microorganism selected from live, killed,
attenuated and inactivated microorganisms. The matrix material may
also comprise a surfactant and may further comprise an adjuvant.
The invention further relates to the manufacture and use of such
compositions, and to other subject matter.
Inventors: |
Coulter; Ivan; (Dublin,
IE) ; McDonald; Bernard Francis; (Castleblayney,
IE) ; Aversa; Vincenzo; (Dublin, IE) ; Rosa;
Monica Torres; (Dublin, IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sigmoid Pharma Limited |
Dublin |
|
IE |
|
|
Assignee: |
Sigmoid Pharma Ltd.
Dublin
IE
|
Family ID: |
46766220 |
Appl. No.: |
14/412420 |
Filed: |
July 5, 2013 |
PCT Filed: |
July 5, 2013 |
PCT NO: |
PCT/EP2013/064327 |
371 Date: |
December 31, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61782066 |
Mar 14, 2013 |
|
|
|
Current U.S.
Class: |
424/486 ;
424/257.1; 424/261.1 |
Current CPC
Class: |
A61K 2039/542 20130101;
A61K 2039/5252 20130101; A61K 2039/5254 20130101; A61K 9/5161
20130101; A61K 39/08 20130101; A61K 2039/522 20130101; A61K 2039/52
20130101; A61K 9/50 20130101; Y02A 50/472 20180101; A61K 9/06
20130101; A61K 2039/521 20130101; A61K 2039/55511 20130101; A61K
9/5042 20130101; A61K 9/5073 20130101; A61K 39/39 20130101; A61K
2039/525 20130101; A61K 39/107 20130101; Y02A 50/30 20180101; A61K
39/0283 20130101; A61K 39/0208 20130101; A61K 39/12 20130101; A61K
39/0258 20130101; A61K 2039/54 20130101; A61K 47/36 20130101; Y02A
50/474 20180101; A61K 9/146 20130101; A61K 39/0002 20130101 |
International
Class: |
A61K 9/06 20060101
A61K009/06; A61K 39/02 20060101 A61K039/02; A61K 39/39 20060101
A61K039/39; A61K 39/108 20060101 A61K039/108 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 5, 2012 |
GB |
1212010.1 |
Claims
1.-69. (canceled)
70. A composition comprising: a matrix comprising a
hydrogel-forming polymer; and comprised in the matrix, a
microorganism selected from live, killed, attenuated and/or
inactivated microorganisms, a surfactant and an adjuvant, wherein
the composition when placed in an aqueous dissolution medium
releases self-assembly structures comprising surfactant; wherein
the self-assembly structures have a hydrodynamic diameter of about
0.5 nm to 200 nm measured using dynamic light scattering.
71. The composition of claim 70 wherein the surfactant is a
non-ionic surfactant.
72. The composition of claim 70 wherein the surfactant is selected
from the group consisting of: macrogol esters; macrogol ethers;
diblock copolymers; triblock copolymers; and amphiphilic polymers;
and combinations thereof.
73. The composition of claim 70 wherein the surfactant comprises
PEGylated hydroxy fatty acid.
74. The composition of claim 70 wherein the surfactant is a
macrogol-15-hydroxystearate.
75. The composition of claim 70 wherein the surfactant is present
in the composition at 20 to 55% by weight, based upon the dry
weight of the composition.
76. The composition of claim 70 wherein at least a portion of the
adjuvant is associated with at least a portion of the
surfactant.
77. The composition of claim 70 wherein at least a portion of the
microorganism content is associated with at least a portion of the
surfactant.
78. The composition of claim 70 wherein the weight ratio of said
surfactant to the hydrogel-forming polymer is from 1:0.5 to
1:2.5.
79. The composition of claim 70 wherein the hydrogel-forming
polymer is selected from the group consisting of gelatin, agar,
agarose, pectin, carrageenan, and chitosan, and combinations
thereof.
80. The composition of claim 70 wherein the adjuvant comprises
.alpha.-galactosylceramide.
81. The composition of claim 70 wherein the composition is
substantially free from oils other than the surfactant.
82. The composition of claim 70 which has a coating selected from
an immediate release coating and a controlled release coating.
83. The composition of claim 70 comprising: a) 25 to 70%
hydrogel-forming polymer; b) 20 to 50% surfactant; c) 1 to 10%
plasticiser; and d) 0.01 to 2% adjuvant; wherein the % are by
weight based on the dry weight of the composition.
84. A composition comprising: a matrix comprising a
hydrogel-forming polymer; and comprised in the matrix, a
microorganism selected from live, killed, attenuated and/or
inactivated microorganisms, a surfactant and an adjuvant; wherein
the adjuvant comprises, a ceramide; and wherein the surfactant
comprises a polyethoxylated hydroxy fatty acid.
85. A process which comprises mixing: i) a surfactant premix
comprising a surfactant, an adjuvant, wherein the adjuvant
comprises a ceramide, and a microorganism selected from live,
killed, attenuated and/or inactivated microorganisms; and ii) a
liquid aqueous premix comprising water and a hydrogel-forming
polymer; and iii) ejecting the mixture of i) and ii) through a
single orifice nozzle to form droplets, the hydrogel-forming
polymer then being caused or allowed to solidify whereby the
droplets form beads; wherein the surfactant premix is at a lower
temperature than the aqueous premix when the two premixes are mixed
together; and wherein following mixing the surfactant is present as
self-assembly structures dispersed within the hydrogel forming
polymer.
86. The process of claim 85 wherein the two premixes are mixed by
an in-line mixing apparatus juxtaposed to the nozzle.
87. A surfactant pre-mix comprising: (i) a surfactant; (ii) a
microorganism selected from live, killed, attenuated and/or
inactivated microorganisms; and (iii) an adjuvant, wherein the
adjuvant is a ceramide.
88. The pre-mix of claim 87, wherein the surfactant comprises a
macrogol-15-hydroxystearate and the ceramide is
.alpha.-galactosylceramide.
89. A method of treatment selected from: i. vaccinating a subject;
ii. induction of an immunotherapeutic response in a subject; or
iii. administration of active entities to induce or regulate
immunity or to locally target metastatic or micrometastatic cells
in the lymphatic system of a subject; the method comprising
administering to the subject a therapeutically effective amount of
the composition of claim 1.
Description
[0001] This invention relates to compositions for delivering one or
more active ingredients, and more particularly to compositions,
e.g. beads, comprising a matrix material which matrix material
comprises a microorganism. In particular, the invention relates to
compositions comprising a microorganism selected from live, killed,
attenuated and inactivated microorganisms. The matrix material may
also comprise a surfactant and may further comprise an adjuvant.
The invention further relates to the manufacture and use of such
compositions, and to other subject matter.
BACKGROUND
[0002] The prior art discloses a modified release dosage product
comprising a plurality of minicapsules (also termed "beads")
containing an active ingredient. The beads may be produced by
mixing two different liquids which are not or are hardly soluble
with each other and one of which is an aqueous liquid comprising
gelatin or another gelling agent. The liquids are mixed and the
resulting mixture is ejected through a nozzle which may have a
single orifice. The nozzle may be vibrated as the mixture is
ejected through it. The ejected mixture forms into drops which are
more or less spherical and fall into a cooling gas (e.g. air) or
into a cooling or hardening solution whereby the gelling agent gels
and the drops become minicapsules. There are disclosed beads made
by ejecting an oil-in-water emulsion whose aqueous phase comprises
gelatin or another water-soluble polymer matrix material through a
single orifice nozzle; the beads include an active agent and, after
drying, can be described as a dried oil-in-water emulsion in which
the dried aqueous phase comprises polymer matrix material. See for
example WO 2004/084870, WO 2008/132712 and WO 2010/133609, all of
which are incorporated herein in their entirety by reference. The
size of the oil droplets of such prior art dried oil-in-water
emulsions is not disclosed in the prior art but has been measured
and found to be around 100 nm, or occasionally down to about 50
nm.
[0003] An X-ray tomography image of the bead of the above
application WO 2010/133609 is shown in FIG. 9. The image
illustrates the highly homogeneous nature of the bead i.e. the near
universal dispersion of the oil phase throughout the aqueous
phase.
[0004] Within the body dendritic cells play a vital role in the
immune system. The main purpose of dendritic cells is to process
antigen material and present the antigen to other cells of the
immune system. Intestinal dendritic cells are found in the
gut-associated lymphoid tissue, including the lamina propria of the
small and large intestine, the isolated lymphoid follicles, the
Peyer patches, and the mesenteric lymph nodes. Dendritic cells
exist in two functionally distinct states: immature and mature
cells. Immature dendritic cells are present in peripheral tissues
and are mainly phagocytic cells; mature dendritic cells are found
in lymphoid organs and are specialized in antigen presentation.
Mature dendritic cells derive from immature cells after a
maturation process that is initiated by inflammatory stimuli and
that leads to a massive migration of dendritic cells to draining
lymph nodes (Banchereau, Nature. 392:245-252; Steinman, Eur. J.
Immunol. 37 S53-S60).
[0005] Several observations in humans and in mouse models of
Inflammatory Bowel Disease suggest that dendritic cells may play a
pathogenic role. Dysfunctional dendritic cells may: act by priming
abnormal responses of T cells to the enteric flora in organized
lymphoid tissues; sustain T cell reactivity within the inflamed
mucosa through the interaction with T cells; and function as
effector cells via the release of proinflammatory cytokines
(Rescigno, J. Clin. Invest. 119:2441-2450).
[0006] Dendritic cells are potent immunostimulatory cells (Steinman
1991) and intestinal dendritic cells actively participate in
antigen capture across the intestinal epithelium by extending
protrusions directly into the lumen for antigen sampling (Rescigno,
Nat. Immunol. 2:361-367). These cells can take up and present both
orally and intestinally administered antigens to naive T cells (Liu
and MacPherson, 1991). Efficient capture and presentation of
antigens by dendritic cells is thought to be central to the
induction of an immune response (Colaco, 1999).
[0007] In the case of a known antigen as seen in celiac disease, a
potential dendritic cell-based, antigen-specific strategy may take
advantage of the ability of dendritic cells to expand and induce
Tregs--the principal effectors of tolerance, which in turn suppress
other dendritic cells that present disease-producing antigens
(Steinman, Immunity. 29:319-324)
BRIEF SUMMARY OF THE DISCLOSURE
[0008] The full ambit of the invention is disclosed in the
following specification and claims. To assist the reader, however,
a brief and non-limiting overview is contained in this paragraph.
The invention provides (amongst other things) beads obtainable by
mixing an aqueous solution of gelatin or another hydrogel-forming
polymer, a surfactant which may be a polyethoxylated fatty acid
(e.g. a polyethoxylated hydroxy fatty acid) or polyethoxylated
fatty alcohol, a microorganism selected from live, killed,
attenuated and inactivated microorganisms, and optionally an
adjuvant. At least a portion of the adjuvant may be associated with
at least a portion of the surfactant. The mix is converted to beads
which are considered to comprise a dispersion of surfactant
self-assembly structures (e.g. micelles) in a hydrogel. The beads
are then dried to result in surfactant self-assembly structures
(e.g. micelles), or precursors to release such structures upon
contact with water, dispersed in a polymer matrix. In any event,
the invention includes dried surfactant-containing beads which
deliver self-assembly structures (e.g. micelles) upon contact with
water, e.g. in an aqueous medium of the GI tract (the aqueous
medium may for example be extracted from a GI tract or
synthetically produced). The products and methods disclosed in this
paragraph are part of the invention and therefore may be claimed,
even though the invention is not at all limited to the subject
matter of this paragraph.
[0009] The invention provides in one aspect a composition which
comprises: (i) a surfactant; and (ii) an active ingredient. The
active ingredient comprises a microorganism selected from live,
killed, attenuated and inactivated microorganisms. The composition
may also comprise an adjuvant. The composition may additionally
comprise one or more excipients selected from hydrogel-forming
polymers, particularly thermotropic hydrogel forming polymers. The
composition may consist essentially of the surfactant and one or
more active ingredients. The composition may consist essentially of
the surfactant, one or more active ingredients and water. The
surfactant may be non-ionic. The surfactant may comprise a
hydrophilic chain and a hydrophobic chain. Also to be mentioned are
ionic, e.g. anionic surfactants.
[0010] The invention includes composition comprising: a matrix
comprising a hydrogel-forming polymer; and comprised in the matrix,
a microorganism selected from live, killed, attenuated and
inactivated microorganisms, a surfactant and an adjuvant.
[0011] The invention provides in a particular embodiment a
composition which comprises: (i) a surfactant; (ii) a microorganism
selected from live, killed, attenuated and inactivated
microorganisms; (iii) an adjuvant; and (iv) a hydrogel-forming
polymer in which the surfactant, the microorganism and the adjuvant
are included; wherein the composition when combined with water is
capable of releasing self-assembly structures (e.g. micelles)
comprising surfactant and adjuvant. Said water may for example be
in the form of gastric, intestinal or colonic fluid or a simulated
form of one of them. It will be recalled that the surfactant may
comprise a hydrophilic chain and a hydrophobic chain.
[0012] For all the compositions disclosed herein, at least a
portion of the adjuvant may be associated with at least a portion
of the surfactant.
[0013] For all the compositions disclosed herein, at least a
portion of the microorganism content may be associated with at
least a portion of the surfactant.
[0014] The invention includes within its scope a composition which
comprises: a hydrogel-forming polymer; self-assembly structures
(e.g. micelles) dispersed in the polymer; and a microorganism. The
hydrogel-forming polymer may be combined with water in a gel state
or in a sol state, or the hydrogel-forming polymer may be dry. As
described further herein, the composition may be coated.
[0015] The microorganism is selected from a live, killed,
attenuated and inactivated microorganism. The microorganism may be
included in the composition, with an adjuvant. In the invention,
the microorganism is immunogenic, for example it contains
(internally or externally) or expresses or releases an antigenic
substance which alone or in combination with an adjuvant may
trigger an immune response when administered to a subject. The
composition, e.g. as mentioned in this paragraph, is for
immunogenic use. In particular, the invention provides a
composition comprising: a matrix comprising a hydrogel forming
polymer; and comprised in the matrix, a microorganism selected from
live, killed, attenuated and inactivated microorganisms, a
surfactant and an adjuvant.
[0016] The surfactant which may be in the form of self-assembly
structures (e.g. micelles) or which is capable of forming
self-assembly structures (e.g. micelles) when combined with water
is sometimes referred to herein as the "self-assembly-forming
surfactant" or "micelle-forming surfactant". (The surfactant may of
course comprise a mixture of surfactant compounds).
[0017] An embodiment of the invention can be described as a dry
hydrogel-forming polymer matrix comprising in the matrix a
microorganism selected from live, killed, attenuated and
inactivated microorganisms, a surfactant and an adjuvant. The dry
hydrogel-forming polymer matrix may have therein a dispersion of
self-assembly structures (e.g. micelles) comprising surfactant and,
optionally, adjuvant. An embodiment of the invention can be
described as a dry self-assembly structure-in-hydrogel dispersion
(e.g. a micelle-in-hydrogel dispersion) wherein the self-assembly
structure-former is, or comprises, surfactant and particularly a
compound which comprises a hydrophilic chain and a hydrophobic
chain. An embodiment of the invention can be described as a dried
self-assembly structure-in-hydrogel dispersion wherein, in some
embodiments, the self-assembly structure-former is, or comprises, a
compound which comprises a hydrophilic chain and a hydrophobic
chain. In one embodiment the composition is not a powder. In other
embodiments the composition is moulded and/or shaped e.g. in the
form of beads e.g. spherical beads, or other shaped units. In
embodiments the composition of the invention comprises multiple
self-assembly structures within a moulded or shaped form e.g. a
bead. It will be understood that the term "spherical" refers to
beads which seem substantially or generally of spherical shape to
the human eye and does not require a sphere to a mathematical
standard. In other words, "spherical" beads as described herein are
generally spheroidal in the sense of resembling or approximating to
a sphere. A population of beads of the disclosure, though, may
contain occasional non-spheroidal beads resulting from the
manufacturing process, and reference herein to e.g. a multiplicity
of beads or a population of beads encompasses such collections of
beads which include not only spherical (spheroidal) beads as
described herein but also non-spherical (i.e. non-spheroidal)
beads.
[0018] The self-assembly structure forming surfactant (e.g.
micelle-forming surfactant) when in a dry composition of the
invention may be described as in the form of pro-self-assembly
structures (e.g. pro-micelles).
[0019] The invention includes not only dry compositions but also
"wet" compositions in which the hydrogel-forming polymer is in the
form of a hydrogel. The invention includes liquids in which the
hydrogel-forming polymer is in combination with water in a liquid
state.
[0020] A benefit of the present invention which is unpredictable
from the prior art is the provision of an effective vaccine
formulation which may be advantageously administered orally. In
particular, the invention provides an improved formulation for the
delivery of a "whole cell" vaccine wherein an antigenic substance
is delivered in the form of a microorganism selected from live,
killed, attenuated and inactivated microorganisms. The inventors
have devised a "whole cell" formulation that, when administered
orally, has been found to induce a potent and antigen-specific
antibody response (as demonstrate by in vivo data herein). The
observed improvement in antibody response following administration
of a "whole cell" formulation according to the invention was
significant when compared to administration of a simple "whole
cell" solution. In addition, and unexpectedly, the observed
improvement in terms of systemic and local intestinal response, was
more significant for "whole cell" formulations than for "sub-unit"
formulations that have been tested, for example a cholera toxin B
subunit formulation (see Comparative Studies in Examples).
[0021] Another benefit of the invention is considered to be derived
from the size of the self-assembly structures formed by the
surfactant. In particular, micelles formed by the micelle-forming
surfactant in an aqueous medium have been observed to be smaller
and present in higher number, than oil droplets obtained following
the teaching of WO 2010/133609. Typically micelles formed according
to the disclosure herein being 10-30 nm. This smaller size gives
rise to a higher surface area than the larger oil droplets of the
prior art and the greater number may provide a higher dispersion
and distribution of micelles following release/formation in an
aqueous medium, such as that found in the GI tract. These features
resulting in turn with better contact with the epithelium and
better absorption. It is also to be mentioned that the surfactant
micelles featured in embodiments of the invention (at least in
terms of micelles released by the compositions in use) provide a
more uniform population in terms of size, i.e. have a lower
polydispersity as regards size. Such small micelle sizes of 10-30
nm (or so) are believed to be inherent to the compositions
described in this specification but it is not mandatory, though it
is an option, that the micelles should, or should predominantly
(e.g. at least 75% of them and optionally at least 80% or at least
90%), have sizes within this range.
[0022] Certain compositions of the invention comprise: a
microorganism selected from live, killed, attenuated and
inactivated microorganisms; and an adjuvant; such compositions
advantageously release both components together. For example,
compositions have been tested comprising enterotoxigenic
Escherichia coli (ETEC), which is a whole cell antigenic substance
of which a portion of the cells may be fragmented, and
.alpha.-GalCer, an adjuvant which is an amphiphilic glycolipid
having both hydrophilic and hydrophobic groups by virtue of
possessing a sugar head and a ceramide part which consists of a
fatty acid and sphingoid chain. The actives ETEC and .alpha.-GalCer
have been administered in a composition considered to be capable of
releasing self-assembly structures, e.g. micelles, when combined
with water, in the form of a composition comprising Kolliphor.RTM.
HS15 as a self-assembly structure-forming agent, e.g.
micelle-forming agent. Similar compositions comprising a whole cell
Vibrio Cholerae and .alpha.-GalCer as adjuvant together with
Kolliphor.RTM. HS15 have also been prepared which are considered
capable of releasing self-assembly structures, eg micelles upon
contact with water. It is believed that that during manufacture
these compositions comprise self-assembly structures (e.g.
comprising Kolliphor.RTM. HS15 as a or the self-assembly
structure-forming surfactant) dispersed in a hydrogel (e.g.
comprising gelatin). These compositions are then dried and
optionally coated prior to storage and subsequent administration.
The compositions have been found to be effectively immunogenic and
immuno-protective. The prior art vaccine formulations as
exemplified in WO 2010/133609, are protein-based and are dried
oil-in-hydrogel emulsions. Without being confined by theory, it is
speculated that the compositions of the invention release both
agents together, enabling the adjuvant to prime the appropriate
immune cells prior to, or at the same time as, contact with the
antigen. Still without being confined by theory, it is considered
that at least a portion of the .alpha.-GalCer associates with at
least a portion of the self-assembly structure-forming surfactant
(for example, Kolliphor.RTM. HS15), e.g. includes itself in the
surfactant envelope of micelles. Also in the context of vaccines,
micelles are believed to be of favourable size for
antigen-presenting cell, such as macrophage, B lymphocyte and
dendritic cell uptake.
[0023] Therefore, an aspect of the invention provides a composition
comprising a matrix comprising a hydrogel-forming polymer; and
comprised in the matrix, a microorganism selected from live,
killed, attenuated and inactivated microorganisms (for example,
ETEC, or for example a V. Cholerae), a surfactant and an adjuvant.
Also provided is a composition comprising [0024] a surfactant (for
example a macrogol-15-hydroxystearate, particularly Kolliphor HS
15), [0025] a microorganism selected from live, killed, attenuated
and inactivated microorganisms (for example ETEC, or for example a
V. Cholerae)) [0026] an adjuvant comprising .alpha.-GalCer, and
[0027] a hydrogel-forming polymer in which the surfactant, the
microorganism and the adjuvant, .alpha.-GalCer, are included and
wherein: the composition when combined with water is capable of
releasing self-assembly structures (e.g. micelles). In particular,
the surfactant which is dispersed in the polymer and which may form
self-assembly structures, for example micelles, when combined with
water, is a macrogol-hydroxy fatty acid and particularly a
macrogol-15-hydroxystearate, e.g. is Kolliphor, particularly
Kolliphor HS 15, More particularly, the microorganism is ETEC. In
another aspect the microorganism is a V. Cholerae.
[0028] The extent of absorption of substances by different parts of
the gastrointestinal tract depends inter alia on the
physico-chemical properties of the substance concerned. Thus,
hydrophobic active agents are better absorbed by the small
intestine than by the colon. Certain microorganisms, such as
particular ETEC strains, over-express, amongst other things,
antigenic substances like CAF/I. Such strains thus have a
hydrophobic surface (see PNAS, Jun. 30 2009, vol. 106, no. 26,
10793-10798; and Infect. Immun, February 2006, p. 1062-1071; for
further information). In particular, the invention contemplates a
composition according to any one of the embodiments described
herein which comprises a microorganism strain which express a
colonisation factor, for example CAF/I. Such a strain may be
selected from Recombinant E. coli, Shigella, Salmonella or V.
Cholerae strains overexpressing major colonization factors CAF/I,
CAF/II, CAF/IV and others including DH5.alpha..lamda.pir;
SY327.alpha..lamda.pir, SM10.lamda.pir, WS-4437A, WS-1858B, A18-34,
A18-34Ap, A18-34ApTp, ACAM2010(pSTREP), E1392/75, E1392/75-2A,
PTL003.
[0029] It is also desirable that formulations containing active
agents, for example the microorganisms described herein, are
protected from degradation in general, including protection from
gastric acid and gastric or intestinal enzymes.
[0030] It is advantageous if the formulation is designed to permit
the coincident release of adjuvant(s) and antigen(s) in a form that
is readily ingested by or interacts in an appropriate manner with
suitable immune cells that are at the surface of or lie beneath the
gastrointestinal epithelial barrier.
[0031] The hydrogel-forming polymer matrix (which may be referred
to as the aqueous phase of a dry dispersion) comprises, in one
embodiment, a cross-linked hydrogel-forming polymer e.g. resulting
from chemical or physico-chemical (e.g. drying) solidification of a
fluid aqueous continuous phase such that, in the matrix or dry
micelle dispersion, water is substantially absent and the micelles
are immobilized. In this embodiment, the dry aqueous phase can
therefore be referred to as an immobilization matrix.
[0032] The surfactant phase may optionally comprise, or be, a
surfactant comprising a hydrophobic chain and a hydrophilic chain.
Optionally, the surfactant phase may comprise an active ingredient
(e.g. a hydrophobic active ingredient, an amphiphilic active
ingredient, or both). The surfactant phase may comprise a
hydrophobic excipient, optionally as well as an active ingredient.
In some embodiments, the surfactant phase comprises an amphiphilic
excipient, optionally as well as one or both of a hydrophobic
excipient and an active agent.
[0033] The term "released" in relation to the self-assembly
structures (e.g. micelles) means free to move, egress, coalesce,
dissolve, (re)emulsify etc. although actual movement, egression,
coalescence, association or (re)emulsification is not a requirement
i.e. may not occur and indeed may intentionally be constrained e.g.
by presence of a coat or coating and/or by incorporation of certain
constraining or retarding substances into the hydrogel-forming
polymer matrix.
[0034] The term "self-assembly structure" refers to any type of
micelle, vesicle, microemulsion, lyotropic phase, laminar or other
self-organised structure that forms spontaneously in the presence
of an aqueous environment, or combination thereof. As is known,
such self-assembly structures form when a self-assembly
structure-forming substance, e.g. comprising or consisting of a
surfactant, is present above a certain critical concentration. The
term includes, for example, micelles, inverted micelles and
liposomes, and combinations thereof. The self-assembly structures
referred to in this specification may comprise, or be, micelles.
More information on self-assembly structures can be found in
"Dynamics of Surfactant Self-assemblies Micelles, Microemulsions,
Vesicles and Lyotropic Phases" by Raoul Zana, particularly Chapter
1, all of which is incorporated herein by reference. The release of
self-assembly structures from a bead or other composition may be
determined by contacting the composition with water and observing
for such structures using a suitable analytical method such as
dynamic light scattering.
[0035] In certain embodiments the self-assembly structures are
present as a microemulsion. For example a microemulsion may be
formed during the preparation of the composition according to the
invention upon mixing of the surfactant (optionally together with
the microorganism and/or adjuvant) together with an aqueous phase
comprising the hydrogel-forming polymer. A microemulsion may also
be formed upon release or formation of self-assembly structures
from the composition when the composition is exposed to a
dissolution medium, suitably an aqueous medium, for example a
gastro-intestinal fluid following oral administration of the
composition. A Microemulsion differs from a conventional emulsion
in that the microemulsion is a thermodynamically stable system
comprising very small self-assembly structures such that the
composition has the appearance of a transparent solution (i.e. is
optically isotropic). Reference to a microemulsion is intended to
encompass a system wherein the self-assembly structures are present
in a form such that the composition has the properties of a
microemulsion; namely a thermodynamically stable dispersion of the
self-assembly structures which are sufficiently small to provide an
optically isotropic (transparent) solution. Such structures
comprise the surfactant and may further comprise, for example the
adjuvant.
[0036] Generally the size of the self-assembly structures present
in or which are formed and released from the composition is of the
order of about 0.5 nm to 200 nm for example about 1 nm to 50 nm, or
about 5 nm to 25 nm. The size of the self-assembly structures
present in the composition, or which are formed and/or released
when the composition is exposed to an aqueous dissolution medium
may be measured using known techniques such as photon correlation
spectroscopy, dynamic light scattering or NMR techniques. The
optical isotropicity of a composition, for example a microemulsion
or may be determined using optical methods such as polarisation
microscopy. These and other methods for the characterisation of
microemulsions are well known (see for example Narang et al Int J.
Pharmaceutics 345 (2007) 9-25). A particular method for measuring
the self-assembly structures (including for example micelles) is
dynamic light scattering.
[0037] A further feature of the invention includes a composition
described herein wherein the composition when placed in an aqueous
dissolution medium releases self-assembly structures (for example
micelles) comprising surfactant when water from the dissolution
medium contacts the hydrogel polymer; wherein the self-assembly
structures have a hydrodynamic diameter of about 0.5 nm to 200 nm
for example about 1 nm to 50 nm, or about 5 nm to 25 nm measured
using dynamic light scattering. Suitable dynamic light scattering
apparatus for measuring the size of self-assembly structures are
well known and include for example a Zetasizer Nano S (ex Malvern
Instruments Ltd). The Nano S contains a 4 mW He--Ne laser operating
at a wavelength of 633 nm and an avalanche photodiode (APD)
detector. Reference to the self-assembly structure (for example
micelle) size using dynamic light scattering is a reference to the
hydrodynamic diameter corresponding to the measured diffusion
coefficient. Reference to the water contacting the dissolution
medium refers to water penetrating, for example, any coatings such
as control release or enteric coatings which may be present on the
composition. The nature of the aqueous dissolution medium is not
thought to be critical and may be, for example, water at pH 7.3 to
7.4 and 37.degree. C. Alternatively as described hereafter the pH
may be increased to for example pH 5.5 or above to dissolve an
enteric coating present on the composition and thus enable water to
contact the hydrogel polymer. Alternatively the dissolution medium
may be a gastrointestinal or simulated gastrointestinal fluid. As
described herein the self-assembly structure comprises the
surfactant (or mixture of surfactants) and may further comprise
adjuvant associated with the structure.
[0038] This method may also be used to measure the size of
self-assembly structures such as micelles or other structures
formed during the preparation of the compositions according to the
invention. For example the method may be used measure the size of
the disperse phase when a microemulsion or microemulsion like
composition is formed as a result of mixing the surfactant phase
with the aqueous hydrogel-forming polymer as described herein.
[0039] Certain embodiments comprise a microorganism selected from
live, killed, attenuated and inactivated microorganisms and an
adjuvant. Preferably the adjuvant is a direct- or indirect
immunostimulant e.g. is an immune cell activator, for example an
antigen-presenting cell activator and/or a T-cell activator. A
representative antigen is enterotoxigenic Escherichia coli (ETEC).
A representative and preferred example of an adjuvant is
.alpha.-galactosylceramide (.alpha.-GalCer) (e.g. KRN 7000) or
another glycolipid adjuvant, for example another glycosylceramide
other than .alpha.-GalCer or a glycosylceramide analogue.
Glycosylceramide analogues are well known and described herein, for
example the analogues taught in WO 2004/028475, which is in its
entirety incorporated herein by reference.
.alpha.-Galactosylceramide is particularly preferred.
[0040] Considering now the self-assembly structure-forming
surfactant, it may in particular be a non-ionic surfactant and in
many embodiments comprises a PEG moiety, PEG also being known as
poly(oxyethylene). The surfactant may comprise a hydrophobic chain
selected from alkyl and alkenyl chains; the hydrophobic chain may
be substituted, for example mono-substituted by e.g. hydroxy,
provided that its hydrophobic character is maintained. In certain
embodiments the self-assembly structure-forming surfactant is
selected from the group consisting of: macrogol esters; macrogol
ethers; diblock copolymers; triblock copolymers; and amphiphilic
polymers, and combinations thereof. Preferably the surfactant is
chosen from macrogol esters.
[0041] In certain embodiments the surfactant has a high
hydrophobic-lipophilic balance (HLB), for example the surfactant
has a HLB value of 10 or above, for example from 10 to 15, suitably
from 10 to 14, for example from 12 to 14.
[0042] In certain embodiments, the surfactant may have a wax-like
character. The surfactant may comprise polyglycol esters of fatty
acids, for example polyglycol mono- and di-esters of fatty acids
(for example stearic acid and/or 12-hydroxy stearic acid). In one
embodiment the fatty acid is saturated (for example stearic acid
and/or 12-hydroxy stearic acid). In another embodiment the fatty
acid is unsaturated or a mixture of saturated and unsaturated fatty
acids. In particular, the surfactant may comprise polyoxyethylene
esters of fatty acid, suitably saturated fatty acid (for example
stearic acid and/or 12-hydroxystearic acid). In such embodiments, a
small part of the 12-hydroxy group can be etherified with
polyethylene glycol. The surfactant may also comprise free
polyethylene glycol.
[0043] In certain embodiments the surfactant is the macrogol ester
macrogol-15-hydroxystearate. A representative
macrogol-15-hydroxystearate is marketed as Kolliphor.RTM. HS 15 by
BASF, which conforms to the requirements of the European
Pharmacopoeia monograph number 2052 Macrogol-15-hydroxystearat,
published in the 6.sup.th Edition, July 2006. A particular class of
surfactants useful in the invention are therefore those which
conform to the requirements of the European Pharmacopoeia monograph
number 2052 Macrogol-15-hydroxystearat, published in the 6.sup.th
Edition, July 2006. Reference herein to "Kolliphor" includes
reference to Kolliphor HS 15. Kolliphor HS 15 may be replaced by
another surfactant meeting the requirements of said monograph
number 2052.
[0044] The hydrogel-forming polymer may be a thermotropic
hydrogel-forming polymer, or a combination of such polymers. The
hydrogel-forming polymer may be gelatin.
[0045] Further provided is a self-assembly structure dispersion
(and in particular a micelle dispersion) for use in manufacturing
the composition of the invention, the self-assembly structure
dispersion comprising a surfactant dispersed in an aqueous phase
which comprises a liquid comprising water and a hydrogel-forming
polymer. The dispersion may comprise an antigen comprising a
microorganism selected from live, killed, attenuated and
inactivated microorganisms. The dispersion may also comprise
hydrophobic and/or amphiphilic active ingredients, for example, an
adjuvant and particularly the adjuvant .alpha.-GalCer. The
dispersion may comprise a hydrophilic active ingredient. It will be
appreciated that the self-assembly structure dispersion may have
the same constituents as the composition of the invention except
that the self-assembly structure dispersion additionally contains a
significant amount of water.
[0046] In another aspect, the invention provides a process for
manufacturing a surfactant/active (e.g. microorganism) premix. A
process of the invention comprises mixing a surfactant, an antigen
comprising a microorganism selected from live, killed, attenuated
and inactivated microorganisms, and optionally an adjuvant and
particularly the adjuvant .alpha.-GalCer. The surfactant and the
antigen may be as further described elsewhere herein.
[0047] The invention includes the following process and
compositions obtainable by (having the characteristics of a
composition obtained by) the process, whether directly or
indirectly. The process comprises mixing: [0048] i) a surfactant
premix comprising a surfactant, an adjuvant and a microorganism
selected from live, killed, attenuated and inactivated
microorganisms; and [0049] ii) a liquid aqueous premix comprising
water and a hydrogel-forming polymer.
[0050] The process of the preceding paragraph may further comprises
ejecting the mixture of i) and ii) through a single orifice nozzle
to form droplets, the hydrogel-forming polymer then being caused or
allowed to solidify whereby the droplets form beads. The
hydrogel-forming polymer is a thermotropic polymer or a mixture of
thermotropic polymers, and the aqueous premix is at an elevated
temperature and the surfactant premix is at a temperature not
exceeding ambient temperature, the two premixes flowing through
respective feed lines to a mixing apparatus where the two premixes
are mixed, and wherein at least one of the two premixes travels a
greater distance through its feedline than the mixture does in
travelling from the mixing apparatus to the nozzle. The two
premixes may be mixed in-line at a location juxtaposed to the
nozzle, e.g. by in-line mixing apparatus juxtaposed to the
nozzle.
[0051] The invention further provides a process which comprises:
[0052] (i) mixing materials comprising water, a hydrogel-forming
polymer, a surfactant, and an active ingredient (for example a
microorganism selected from live, killed, attenuated and
inactivated microorganisms; and optionally an adjuvant) to form a
self-assembly structure dispersion (possibly a micelle dispersion)
within an aqueous phase comprising the hydrogel-forming polymer,
the process optionally further comprising [0053] (ii) formulating
the dispersion of (i) into a suitable form, e.g. a bead, by
ejecting it through a single orifice nozzle to form droplets which
are caused or allowed to pass into a cooling medium, e.g. a
water-immiscible cooling liquid, in which the droplets cool to form
shaped units e.g. beads. The invention includes shaped units (e.g.
beads) obtainable by the process, whether directly or indirectly,
i.e. includes shaped units (e.g. beads) having the characteristics
of shaped units obtained by the process, whether directly or
indirectly
[0054] In embodiments, the invention includes a process for
manufacturing a composition or dispersion of the invention which
comprises: forming an aqueous premix which comprises water and
water soluble/dispersible materials and a surfactant premix
(optionally containing an oil) which comprises surfactant and
surfactant soluble/dispersible materials, and combining the two
premixes to form a self-assembly structure dispersion (possibly a
micelle dispersion) within an aqueous phase comprising the
hydrogel-forming polymer. The dispersion may then be formed into a
bead as described in the preceding paragraph. More particularly the
manufacture of the composition may optionally comprise:
[0055] (i) forming an aqueous phase premix comprising, or usually
consisting of, a solution in water of water-soluble constituents
(e.g. hydrogel-forming polymer, any water-soluble excipient(s), any
hydrophilic active(s) (for example, a microorganism could be
included in the aqueous phase premix as, for example an aqueous
suspension);
[0056] (ii) forming a surfactant phase premix comprising, or
usually consisting of, a solution in a surfactant of hydrophobic
and amphiphilic constituents (e.g. active ingredient(s) selected
from a microorganism could be included directly in the surfactant
premix or included as an aqueous suspension in the surfactant
premix for example in the form of a water-in-surfactant
emulsion);
[0057] (iii) mixing the two phases to form a dispersion; and
optionally
[0058] (iv) formulating the dispersion into a bead or other shaped
unit, e.g. ejecting it through a single orifice nozzle to form
droplets which are caused or allowed to fall into a water
immiscible cooling liquid in which the droplets cool to form beads,
and then separating the beads from the cooling liquid.
[0059] Further provided by the invention is a process which
comprises mixing (i) a surfactant, and (ii) an active ingredient
selected from a microorganism; the mixing may further comprise
mixing a hydrophobic excipient, for example a medium chain
triglyceride, and the surfactant and the microorganism. The
resultant surfactant mix may be mixed with an aqueous composition
comprising water and a hydrogel-forming polymer, the surfactant
being in an amount sufficient to form self-assembly structures
(e.g. micelles), the mixing thereby forming a self-assembly
structure dispersion.
[0060] Another process of the invention is a process which
comprises mixing materials comprising (i) water, (ii) a
hydrogel-forming polymer, (iii) a surfactant, and (iv) an active
ingredient, to form a self-assembly structure (e.g. micelle)
dispersion within an aqueous phase comprising the hydrogel-forming
polymer.
[0061] As an intermediate product obtained during manufacture of
the final compositions of the disclosure, the invention includes a
composition comprising a hydrogel having dispersed therein
self-assembly structures (e.g. micelles) comprising a self-assembly
structure-forming compound, the compound optionally being selected
from compounds having a hydrophilic chain and a hydrophobic chain,
the composition further comprising an active agent as described
herein.
[0062] The invention further provides a product having the
characteristics of a composition obtained by drying a composition
comprising a hydrogel having dispersed therein self-assembly
structure (e.g. micelles) comprising a self-assembly structure
(e.g. micelle)-forming compound, the compound optionally being
selected from compounds having a hydrophilic chain and a
hydrophobic chain, the composition further comprising an active
agent.
[0063] It may also be advantageous, e.g. from a manufacturing
perspective, to include an oil with the surfactant in the processes
described herein. The surfactant phase may therefore additionally
include an oil in the process and the surfactant in the product may
be associated with an oil.
[0064] Any pharmaceutically suitable oil or oil acceptable for food
use (or other chosen application) may be used as the oil. In terms
of dry weight of the composition of the invention, the oil may
comprise a proportion from 1% to 85%, e.g. 1% to 50%, optionally 1%
to 30%, 1% to 20%, 1% to 10% or 1% to 5%, The oil may comprise 5%
to 30%, 5% to 20% or 5% to 10%; it may comprise from 20% to 30% or
from 35% to 45%. The term "oil" means any substance that is wholly
or partially liquid at ambient temperature or close-to-ambient
temperature e.g. between 10.degree. C. and 40.degree. C. or between
15.degree. C. and 35.degree. C., for example liquid at a
temperature of up to 25.degree. C. and whether or not liquid within
the entirety of the aforesaid ranges, and which is hydrophobic but
soluble in at least one organic solvent. Oils include vegetable
oils (e.g. neem oil), petrochemical oils, and volatile essential
oils.
[0065] As oils which may be included may be mentioned
poly-unsaturated fatty acids such as, for example, omega-3 oils for
example eicosapentanoic acid (EPA), docosohexaenoic acid (DHA),
alpha-linoleic acid (ALA), conjugated linoleic acid (CLA).
Preferably ultrapure EPA, DHA or ALA or CLA are used e.g. purity up
to or above 98%. Omega oils may be sourced e.g. from any
appropriate plant e.g. sacha inchi. Such oils may be used singly
e.g. EPA or DHA or ALA or CLA or in any combination. Combinations
of such components including binary, tertiary etc combinations in
any ratio are also contemplated e.g. a binary mixture of EPA and
DHA in a ratio of 1:5 available commercially under the trade name
Epax 6000.
[0066] Oils which may be included comprise, or are, particularly
natural triglyceride-based oils which include olive oil, sesame
oil, coconut oil, palm kernel oil. Oils which are particularly
preferred include saturated coconut and palm kernel oil-derived
caprylic and capric fatty acids and glycerin e.g. as supplied under
the trade name Miglyol.TM. a range of which are available and from
which one or more components of the oil phase of the invention may
be selected including Miglyol.TM. 810, 812 (caprylic/capric
triglyceride); Miglyol.TM. 818: (caprylic/capric/linoleic
triglyceride); Miglyol.TM. 829: (caprylic/capric/succinic
triglyceride; Miglyol.TM. 840: (propylene glycol
dicaprylate/dicaprate). Note that Miglyol.TM. 810/812 differ only
in C.sub.8/C.sub.10-ratio and, because of its low C.sub.10-content,
the viscosity and cloud point of Miglyol.TM. 810 are lower. The
Miglyol.TM. range is available commercially from Sasol Industries.
As noted above, oils which may be included in the oil phase need
not necessarily be liquid or fully liquid at room temperature.
Alternative or additional oils which may be included in the oil
phase according to the invention are medium chain triglycerides
such as for example Labrafac.TM. Lipophile manufactured by
Gattefosse in particular product number WL1349.
[0067] Other possible (alternative or additional) oils include
linoleoyl macrogolglycerides (polyoxylglycerides) such as, for
example, Labrafil (e.g. product number M2125CS by Gattefosse) and
caprylocaproyl macrogolglycerides such as, for example, Labrasol by
Gattefosse.
[0068] In other embodiments the surfactant phase/mix used in the
preparation of the compositions is free or substantially free from
other oils. In such embodiments the surfactant phase/mix comprises
(i) a surfactant, (ii) an active ingredient (for example a
microorganism selected from live, killed, attenuated and
inactivated microorganisms; and optionally an adjuvant) is
substantially free or free from other oils, for example the
surfactant phase/mix contains less than 10%, 5%, 1%, 0.5%, 0.1% or
0.001% by weight of oil or is free from other oils. The surfactant
phase/mix may then be mixed with an aqueous composition comprising
water and a hydrogel-forming polymer, as described herein to form a
self-assembly structure dispersion (possibly a micelle dispersion)
within an aqueous phase.
[0069] The beads which have been separated from the cooling liquid
may be centrifuged to remove excess oil and then air-dried, e.g. at
ambient temperature (say 15-30.degree. C., e.g. 20-25.degree. C.).
The centrifuging normally takes place before the air drying.
[0070] As described in more detail later in this specification, a
bead made as described herein may after its preparation be coated
with one or more layers.
[0071] In a further aspect, the present invention provides for a
dosage form comprising a population of optionally coated beads of
the invention. The beads of the dosage form comprise an active
ingredient as described herein. The dosage form is suitable for
pharmaceutical use. In certain embodiments the dosage form may
comprise at least two populations of beads.
[0072] In certain embodiments the dosage form comprises the
composition (e.g. a bead or shaped unit and particularly multiple
beads or shaped units) of the invention in a unit dosage form
suitable for administration, for example to a human or animal. The
unit dosage form chosen from a capsule, a tablet, a sprinkle, a
sachet, a suppository, a pessary or other suitable unit dosage
form.
[0073] In a representative embodiment a dosage form of the
invention is formed by mixing together at least the following
materials to form a self-assembly structure (e.g. micelle)
dispersion: water; a hydrogel-forming polymer; a surfactant; and an
active ingredient, and formulating the dispersion into a dosage
form (suitable for pharmaceutical use) comprising a bead which
comprises the dispersion in a dry state.
[0074] In some embodiments the dosage form has been appropriately
formulated in such a way as to release the one or more active
ingredients at one or more specified locations in the
gastrointestinal tract (GIT). In particular the dosage form is
formulated to release the microorganism and optional adjuvant in at
least the upper small intestine and the dosage form may therefore
be enteric coated. The dosage form may comprise enteric coated
beads, for example the dosage form may be a capsule or other format
comprising a plurality of enteric coated beads. The dosage form may
target release elsewhere e.g. the ileum, the colon or both.
[0075] The dosage forms of the invention are in particular for oral
administration.
[0076] The invention includes a method for administering a
microorganism selected from live, killed, attenuated and
inactivated microorganisms to a subject, comprising orally
administering to the subject a composition or dosage form as
disclosed herein, which composition or dosage form comprises such
an active agent (e.g. a combination of (i) a microorganism selected
from live, killed, attenuated and inactivated microorganisms and
(ii) an adjuvant).
[0077] The invention also includes a method for administering an
active pharmaceutical ingredient comprising a microorganism
selected from live, killed, attenuated and inactivated
microorganisms to a subject, the method comprising administering a
dosage form comprising a population of beads. The beads comprise a
matrix comprising a hydrogel-forming polymer, and comprised in the
matrix, a surfactant, an optional adjuvant and a microorganism
selected from live, killed, attenuated and inactivated
microorganisms. The dosage form is for oral administration. The
dosage form may be adapted to release the active ingredient in the
gastrointestinal tract.
[0078] Provided by the invention also is a product having the
characteristics of a composition obtained by drying a composition
comprising a hydrogel having dispersed therein self-assembly
structures (e.g. micelles), the composition further comprising a
microorganism selected from live, killed, attenuated and
inactivated microorganisms, and optionally an adjuvant and the use
of the product in the manufacture of an oral dosage form, for
example a gelatin capsule.
[0079] Further included in the invention is a process for
administering a microorganism selected from live, killed,
attenuated and inactivated microorganisms to a subject, comprising
orally administering to the subject a product comprising an active
agent (as described herein), wherein the product is a composition
as described herein.
[0080] Also provided by the invention is a composition or dosage
form as disclosed herein, which composition or dosage form
comprises an active agent (as disclosed herein) (e.g. combination
of active agents as disclosed herein) for use as a medicament.
[0081] Also provided by the invention is a method for performing a
treatment selected from:
[0082] i. vaccinating a subject [0083] ii. induction of an
immunotherapeutic response e.g. to treat diseases selected from
cancers and autoimmune diseases (e.g. to control autoimmune
diseases), the autoimmune diseases being selected from systemic and
gastrointestinal autoimmune diseases [0084] iii. administration of
active entities to induce or regulate immunity or to locally target
metastatic or micrometastatic cells in the lymphatic system, the
method comprising administering a composition or dosage form as
disclosed herein, which composition or dosage form comprises an
active agent (as disclosed herein) (e.g. combination of active
agents as disclosed herein) having an activity appropriate to
achieve the recited treatment.
[0085] Also provided by the invention is a composition or dosage
form as disclosed herein, which composition or dosage form
comprises an active agent (as disclosed herein) (e.g. combination
of active agents as disclosed herein) for use in the manufacture of
a medicament for prophylaxis or treatment of a disease or medical
condition described herein, for example a treatment selected from
[0086] i. vaccinating a subject [0087] ii. induction of an
immunotherapeutic response e.g. to treat diseases selected from
cancers and autoimmune diseases (e.g. to control autoimmune
diseases), the autoimmune diseases being selected from systemic and
gastrointestinal autoimmune diseases [0088] iii. administration of
active entities to induce or regulate immunity or to locally target
metastatic or micrometastatic cells in the lymphatic system.
[0089] Throughout the description and claims of this specification,
the words "comprise" and "contain" and variations of them mean
"including but not limited to", and they are not intended to (and
do not) exclude other moieties, additives, components, integers or
steps. Throughout the description and claims of this specification,
the singular encompasses the plural unless the context otherwise
requires. In particular, where the indefinite article is used, the
specification is to be understood as contemplating plurality as
well as singularity, unless the context requires otherwise.
[0090] Features, integers, characteristics, compounds, chemical
moieties or groups described in conjunction with a particular
aspect, embodiment or example of the invention are to be understood
to be applicable to any other aspect, embodiment or example
described herein unless incompatible therewith. All of the features
disclosed in this specification (including any accompanying claims,
abstract and drawings), and/or all of the steps of any method or
process so disclosed, may be combined in any combination, except
combinations where at least some of such features and/or steps are
mutually exclusive. The invention is not restricted to the details
of any foregoing embodiments. The invention extends to any novel
one, or any novel combination, of the features disclosed in this
specification (including any accompanying claims, abstract and
drawings), or to any novel one, or any novel combination, of the
steps of any method or process so disclosed.
[0091] The readers attention is directed to all papers and
documents which are filed concurrently with or previous to this
specification in connection with this application and which are
open to public inspection with this specification, and the contents
of all such papers and documents are incorporated herein by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0092] The trade mark "SmPill" herein refers to a composition of
the invention and more particularly a composition of the invention
as described in the examples.
[0093] FIG. 1. Immunization with ETEC and alpha-GalCer in
SmPill.RTM. induces significantly stronger mucosal antibody titres
than delivery of antigen in solution or in non-adjuvanted
SmPill.RTM.. Mice were immunised on 2 consecutive days on week 0
with ETEC alone or with alpha-GalCer or CT in solution or with ETEC
either alone or with alpha-GalCer in SmPill.RTM., followed by an
identical series of booster immunisations at week 2 and 4. Faecal
pellets were collected 1 day prior to booster immunizations and 12
days post the final immunization and antigen-specific IgA (a) and
IgG (b) antibody titres were determined by ELISA. Arrows represent
booster immunizations. ***, P<0.001 ETEC+alpha-GalCer
SmPill.RTM. vs ETEC+alpha-GalCer solution, +++, P<0.001
ETEC+alpha-GalCer SmPill.RTM. vs ETEC SmPill.RTM..
[0094] FIG. 2. Oral administration of ETEC with alpha-GalCer in
SmPill.RTM. induces potent antigen-specific IgA responses in faecal
pellets. Mice were immunised as described in the legend to FIG. 1.
Faecal pellets were collected 12 days post the final immunization
and antigen-specific IgA titres and total IgA antibody
concentrations were assessed by ELISA. Results are expressed as
CFA/I-specific IgA endpoint titres (a) or antigen-specific IgA
titres/total IgA concentrations (b). +++P<0.001
ETEC+alpha-GalCer SmPill.RTM. vs ETEC solution, *** P<0.001
ETEC+alpha-GalCer SmPill.RTM. vs ETEC SmPill.RTM..
[0095] FIG. 3. Oral immunisation with ETEC and alpha-GalCer in
SmPill.RTM. induces strong antigen-specific systemic antibody
responses. Mice were immunised as described in the legend to FIG.
1. Antigen-specific IgA and IgG antibody responses were assessed by
ELISA on serum samples recovered 1 day prior to booster
immunizations and 12 days post the final immunization. Arrows
represent booster immunizations. **, P<0.01, ***, P<0.001
ETEC+alpha-GalCer SmPill.RTM. vs ETEC+alpha-GalCer solution, ++,
P<0.01, +++, P<0.001 ETEC+alpha-GalCer SmPill.RTM. vs ETEC
SmPill.RTM..
[0096] FIG. 4. Oral administration of ETEC with alpha-GalCer in
SmPill.RTM. induces potent antigen-specific IgA responses
systemically. Mice were immunised as described in the legend to
FIG. 1. Serum samples were collected 12 days post the final
immunization and antigen-specific IgA titres (a) and total IgA
antibody concentrations (b) were assessed by ELISA.
[0097] FIG. 5. Oral immunisation of mice with ETEC and alpha-GalCer
in SmPill.RTM. induces a predominant IgG1 antibody response
systemically. Mice were immunized as described in the legend to
FIG. 1. Antigen-specific IgG1, IgG2a and IgG2b antibody titres were
assessed by ELISA on sera recovered 12 days post the final
immunization. Results are presented as IgG1, IgG2a or IgG2b
end-point titres.
[0098] FIG. 6. Oral immunization of mice with SmPill.RTM.
containing ETEC and alpha-GalCer as adjuvant induces IgG antibody
responses locally in the intestines. Mice were immunized as
described in the legend to FIG. 1. Antigen-specific IgG antibody
titres were determined by ELISA on both small and large intestinal
washes recovered two weeks post the final series of immunizations.
+P<0.05, +++P<0.001 ETEC+alpha-GalCer SmPill.RTM. vs ETEC
solution, ** P<0.01, *** P<0.001 ETEC+alpha-GalCer
SmPill.RTM. vs ETEC SmPill.RTM..
[0099] FIG. 7. Oral immunization of mice with SmPill.RTM.
containing ETEC and alpha-GalCer as adjuvant induces significant
IgA antibody responses locally in the intestines. Mice were
immunized as described in the legend to FIG. 1. Antigen-specific
IgA antibody titres and total IgA antibody concentrations were
determined by ELISA on supernatants obtained from saponin-treatment
of both small and large intestinal extracts recovered two weeks
post the final series of immunizations. Results are expressed as
CFA/I-specific IgA endpoint titres ((a) and (c)) or
antigen-specific IgA titres/total IgA concentrations ((b) and (d)).
+++P<0.001 ETEC+alpha-GalCer SmPill.RTM. vs ETEC solution, ***
P<0.001 ETEC+alpha-GalCer SmPill.RTM. vs ETEC SmPill.RTM..
[0100] FIG. 8. Oral immunization of mice with SmPill.RTM.
containing ETEC and alpha-GalCer as adjuvant induces significant
IgG antibody responses locally in the intestines. Mice were
immunized as described in the legend to FIG. 1. Antigen-specific
IgG antibody titres were determined by ELISA on supernatants
obtained from saponin-treatment of both small and large intestinal
extracts recovered two weeks post the final series of
immunizations. +++P<0.001 ETEC+alpha-GalCer SmPill.RTM. vs ETEC
solution, *** P<0.001 ETEC+alpha-GalCer SmPill.RTM. vs ETEC
SmPill.RTM..
[0101] FIG. 9 shows an x-ray tomography image of the bead that is
described in WO 2010/133609.
[0102] FIG. 10 shows the results of a reference study (1). Oral
administration to mice of Cholera toxin subunit B (CTB) with and
without alpha-GalCer and Kolliphor HS 15 in SmPill.RTM. induces
antigen-specific IgG responses systemically. Mice were immunised
analogously to the legend to FIG. 1. Serum samples were collected
12 days post the final immunization and antigen-specific IgG titres
(a) were assessed by ELISA.
[0103] FIG. 11 shows the results of a reference study (1). Oral
administration to mice of Cholera toxin subunit B (CTB) with and
without alpha-GalCer and Kolliphor HS 15 in SmPill.RTM. induces
antigen-specific IgG responses systemically. Mice were immunised
analogously to the legend to FIG. 1. Serum samples were collected
12 days post the final immunization and antigen-specific IgA titres
(a) and total IgA antibody concentrations (b) were assessed by
ELISA.
[0104] FIG. 12 shows the results of a reference study (2). Oral
administration to mice of Cholera toxin subunit B (CTB) with and
without alpha-GalCer and Kolliphor HS 15 in SmPill.RTM. induces
antigen-specific IgG and IgA responses locally in the intestines.
Mice were immunized analogously to the legend to FIG. 1.
[0105] FIG. 13 Oral immunisation of mice with SmPill.RTM.
formulations of JS1569 V. Cholerae bacteria adjuvanted with
.alpha.GalCer induce strong fecal pellet antibody responses in
mice. Mice were immunized as described in V. Cholerae Experiment 1
and fecal pellets were analysed. FIG. 13 the IgA titres observed in
fecal pellets (a) after 1 round, (b) after 2 rounds and (c) after 3
rounds of immunisation; (e) shows the IgG titre in fecal pellets
after 3 rounds of immunisation which were measured by ELISA.
Results in FIG. 13 represent the mean and SD of all mice in each
group.
[0106] FIG. 14 Shows a time course illustrating the kinetics of the
induction of the IgA response after each round of immunisation
described in FIG. 13.
[0107] FIG. 15 Oral immunisation of mice with SmPill.RTM.
formulations of JS1569 V. Cholerae bacteria adjuvanted with
.alpha.GalCer induce stronger tissue specific antibody responses
versus delivery in solution. Mice were orally vaccinated as
described in V. Cholera Experiment 1. 2 weeks after the final round
of vaccination mice were euthanized and perfused to remove the
blood from the internal organs. FIG. 15 shows the Inaba specific
LPS IgA (a) and IgG (b) titres measured by ELISA on the small
intestinal washes. Results represent the mean and SD of all mice in
each group
[0108] FIG. 16: Oral immunisation of mice with SmPill.RTM.
formulations of JS1569 V. Cholerae bacteria adjuvanted with
.alpha.GalCer induce stronger serum antibody responses than
identical formulations delivered in solution. Mice were orally
vaccinated as described in V. Cholerae Experiment 1. 2 weeks after
the final round of vaccination serum was obtained by tail bleeds.
FIG. 16 shows Inaba specific LPS IgA (a), IgG (b) and IgG1 (c)
titres measured by ELISA. Results represent the mean and SD of all
mice in each group.
[0109] FIG. 17 Higher SmPill.RTM. doses of JS1569 V. Cholerae
bacteria with .alpha.GalCer induce fecal IgA responses in mice.
Mice were orally vaccinated as described in V. Cholerae Experiment
4. Fecal pellet samples were obtained the day before each round of
vaccination and 2 weeks after the final vaccination and anti
Inaba-LPS IgA and IgG responses measure. FIG. 17 shows the various
IgA titres observed after 1, 2 and 3 rounds of immunisation which
were measured by ELISA. Results represent OD492 absorbance values
over a series of dilutions of the sample.
[0110] FIG. 18 Vaccinating with higher doses of SmPill.RTM. V.
Cholerae JS1569 bacteria adjuvanted with .alpha.GalCer Induces
tissue specific IgA responses in mice. Mice were orally vaccinated
as described in V. Cholerae Experiment 4. 2 weeks after the final
round of vaccination mice were euthanized and perfused to remove
the blood from the internal organs. The small intestine was
dissected out and a small portion of the upper section removed that
placed into a saponin solution to extract the inter tissue
antibodies. FIG. 18 show the various Inaba LPS-specific IgA titres
observed after 3 rounds of immunisation which were measured by
ELISA. Results represent OD492 absorbance values over a series of
dilutions of the sample.
DETAILED DESCRIPTION
[0111] The term "amphiphilic" means the same as "amphipathic" and
means containing both a hydrophilic group and a hydrophobic
(lipophilic) group.
[0112] The term "associated with" includes reference to two
substances being mixed or having an interface with each other. For
example, where an adjuvant is associated with a surfactant in a
bead or other dried composition, the association may be determined
by identifying co-location of the adjuvant and the active using a
suitable analytical technique. (Co-location means the existence of
at least one location in which both substances are located).
Analytical techniques to determine co-location may include TOFF,
mapping Raman spectroscopy and infrared spectroscopy. A composition
wherein at least a portion of the adjuvant is associated with at
least a portion of the surfactant may therefore be a composition
comprising location(s) at which adjuvant and surfactant are
co-located; all the adjuvant (e.g. substantially all the adjuvant)
may be co-located with surfactant.
[0113] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are generally regarded as safe. In
particular, pharmaceutically acceptable carriers used in the
practice of this invention are physiologically tolerable and do not
typically produce an allergic or similar untoward reaction (for
example, gastric upset, dizziness and the like) when administered
to a patient. Preferably, as used herein, the term
"pharmaceutically acceptable" means approved by a regulatory agency
of the appropriate governmental agency or listed in the U.S.
Pharmacopoeia or other generally recognized pharmacopoeia for use
in animals, and more particularly in humans.
[0114] A "pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes an excipient that is acceptable for
veterinary use as well as human pharmaceutical use. A
"pharmaceutically acceptable excipient" as used in the present
application includes both one and more than one such excipient.
[0115] The term "release", particularly in relation to
self-assembly structure (e.g. micelles), includes reference both to
releasing pre-existing self-assembly structures in a polymer matrix
and to release self-assembly structures comprising surfactant not
in self-assembly structure form in the polymer matrix but formed
after ingestion of a composition of the invention as water and the
self-assembly structure come into mutual contact. In other words a
self-assembly structure released from a composition of the
invention may be preformed in the composition or formed (in whole
or in part) as part of the release process.
[0116] The term "treating" includes: (1) preventing or delaying the
appearance of clinical symptoms of the state, disorder or condition
developing in an animal that may be afflicted with or predisposed
to the state, disorder or condition but does not yet experience or
display clinical or subclinical symptoms of the state, disorder or
condition; (2) inhibiting the state, disorder or condition (e.g.,
arresting, reducing or delaying the development of the disease, or
a relapse thereof in case of maintenance treatment, of at least one
clinical or subclinical symptom thereof); and/or (3) relieving the
condition (i.e., causing regression of the state, disorder or
condition or at least one of its clinical or subclinical symptoms).
The benefit to a patient to be treated may be either statistically
significant or at least perceptible to the patient or to the
physician. The term "therapeutic or prophylactic" encompasses the
same subject matter.
[0117] "Vaccine" is herein defined as a composition comprising an
antigenic substance, in particular comprising modified-live (live
attenuated) or inactivated infectious agent or microorganism, that
is administered, most often with an adjuvant, into an animal to
produce an immunologically mediated effect such as active immunity,
induction of tolerance, breaking of tolerance, altering the course
of an auto-immune disease etc. The composition of the invention may
be a vaccine composition. Unless the context so demands, the term
"vaccine composition" includes immunomodulation which is not
necessarily vaccination e.g. toleration or other immunotherapy.
[0118] The term "subject" includes birds, humans and other mammals
as well as fish, for example domestic animals (e.g., dogs and
cats). The term "subject" in particular denotes a human.
[0119] "Effective amount" means an amount sufficient to result in
the desired therapeutic or prophylactic response. The therapeutic
or prophylactic response can be any response that a user (e.g., a
clinician) will recognize as an effective response to the therapy.
It is further within the skill of one of ordinary skill in the art
to determine appropriate treatment duration, appropriate doses, and
any potential combination treatments, based upon an evaluation of
therapeutic or prophylactic response.
[0120] The terms "dry" and "dried" as applied to compositions of
the disclosure may each include reference to compositions
containing less than 5% free water by weight, e.g. less than 1%
free water by weight. Primarily, however, "dry" and "dried" as
applied to compositions of the disclosure mean that the hydrogel
present in the initial solidified composition has dried
sufficiently to form a rigid composition.
[0121] As previously described the invention provides amongst other
things a composition comprising: (i) a hydrogel-forming polymer;
(ii) a surfactant; and (iii) a microorganism selected from live,
killed, attenuated and inactivated microorganisms, the composition
further comprising a feature selected from: (i) at least a portion
of the surfactant is in the form of self-assembly structures (e.g.
micelles) dispersed in the polymer; and (ii) the composition is
capable of releasing self-assembly structures (e.g. micelles) when
combined with water. The composition may also comprise an adjuvant.
Said water may for example be in the form of gastric, intestinal or
colonic fluid or a simulated form of one of them. The invention
also provides a composition comprising a (i) hydrogel having
dispersed therein self-assembly structure (e.g. micelles)
comprising a self-assembly structure-former (e.g. micelle-former),
e.g. a compound selected from surfactants comprising a hydrophilic
chain and a hydrophobic chain, and (ii) a microorganism selected
from live, killed, attenuated and inactivated microorganisms, the
drying of such compositions and the dried compositions. The
composition may also comprise an adjuvant.
[0122] The invention will now be described in detail by reference
to the various components which the composition of the invention
may comprise. The term "excipient" may be used occasionally to
describe all or some of the components other than the active
ingredient(s) bearing in mind that some excipients can be active
and that some active principles can have excipient character.
[0123] If not otherwise stated, ingredients, components, excipients
etc of the composition of the invention are suitable for one or
more of the intended purposes discussed elsewhere herein e.g. are
cosmetically acceptable, environmentally acceptable,
pharmaceutically acceptable, acceptable as food additives etc.
[0124] For the avoidance of doubt, it is hereby stated that the
information disclosed earlier in this specification under the
heading "Background" is relevant to the invention and is to be read
as part of the disclosure of the invention.
Active Ingredient
[0125] The compositions of the invention comprise a microorganism
as an active ingredient. In the invention, the microorganism is
selected from live, killed, attenuated and inactivated
microorganisms. The composition may also comprise an adjuvant as an
active ingredient. It is preferred that the invention comprise a
bead which comprises, as active ingredients, a microorganism
selected from live, killed, attenuated and inactivated
microorganisms and an adjuvant.
[0126] The microorganism may be a unicellular microorganism, for
example it may be selected from bacteria, unicellular fungi and
protozoa. The microorganism may be an intestinal pathogen. The
microorganism may be a pathogen, e.g. an intestinal pathogen, of a
bird or mammal, e.g. human. The microorganism may be a human
intestinal pathogen. The microorganism may be a bacterium
expressing colonization factor antigen I (CAF/I), and may be a
human pathogen expressing CAF/I. The microorganism may comprise a
combination of microorganisms, e.g. the microorganisms being as
described earlier in this paragraph, for example a combination of
microorganisms expressing CAF/I.
[0127] The microorganisms may be killed, attenuated or inactivated
by any means known to the skilled person including, for example,
radioactivity, e.g. non-ionising radiation, ionising radiation,
gamma radiation or infrared radiation; ultrasonic vibrations; heat
inactivation e.g. moist heating or dry heating; chemical
inactivation, e.g. the use of formalin, an alcohol, a phenol,
ethylene oxide, propiolactone, ethyleneimine or carbon dioxide; the
use of an antibiotic; a physio-chemical method, e.g.
steam-formaldehyde; attenuation through a foreign host, e.g. tissue
culture, embryonated eggs or live animals, or a combination
thereof. It is to be understood that, during the process of
killing, attenuating or inactivating, or indeed during the
formulation process, the live, killed, attenuated or inactivated
microorganisms may be partially fragmented. The skilled person will
therefore understand that the use of the term live, killed,
attenuated and inactivated microorganisms may refer to a mixture of
unfragmented (intact) and fragmented microorganism units; the
microorganisms may be intact, although it will be understood that
for practical a population of intact microorganisms may include a
small proportion of fragmented microorganisms and the term "intact
microorganism" is to be construed accordingly.
[0128] The microorganism may be formalin-killed. It is expected
that formalin-killed microorganisms may be wholly, partly or
predominantly fragmented, and the term "killed microorganism"
therefore includes within its scope intact dead microorganisms and
fragmented dead microorganisms and combinations thereof.
[0129] It will be understood that the term "fragmented
microorganism" refers to a product obtainable by (having the
characteristics of a product obtained by) fragmentation of an
intact microorganism, and is therefore to be distinguished from,
and does not include, purified or isolated subunits or fragments of
microorganisms, as these do not include the residue of
(substantially) an entire microorganism.
[0130] A hydrophobic active ingredient is determined by the
compound being partially or fully soluble in non-aqueous medium and
insoluble in aqueous medium. The hydrophobic active ingredient is
partially or fully soluble in a non-aqueous environment.
[0131] An amphiphilic active ingredient is determined by the
presence of both hydrophilic and hydrophobic regions in the
compound. The active ingredient is therefore partially or fully
soluble in both an aqueous medium and a non-aqueous medium.
[0132] In certain embodiments the composition may comprise a
hydrophilic active ingredient. In embodiments the composition may
comprise a further active ingredient, the further active ingredient
being a hydrophilic active ingredient. A hydrophilic active
ingredient is partially or fully soluble in an aqueous medium and
insoluble in non-aqueous medium.
[0133] Compositions of the invention therefore comprise one or more
antigens comprising a microorganism selected from live, killed,
attenuated and inactivated microorganisms. Additionally, such
compositions may comprise an adjuvant, whether a single adjuvant or
a combination thereof; for example there may be used as an adjuvant
a glycolipid adjuvant such as, for example, .alpha.-GalCer or an
analogue thereof. "Antigen" is herein defined to include reference
to a substance or compound which, when introduced into a non-human
animal or a human, will result in the formation of antibodies
against the antigen and/or cell-mediated immunity; the antigen
content of the compositions of the disclosure comprises or consists
of a microorganism, e.g. a single microorganism or a combination of
microorganisms, selected from live, killed, attenuated and
inactivated microorganisms. As described elsewhere herein, the
microorganism is selected from intact and fragmented microorganisms
and combinations thereof.
[0134] Antigens are commercially available or one of skill in the
art is capable of producing them. The one or more antigenic
moieties comprised in the vaccine comprise, for example, either a
modified-live or killed microorganism (e.g. chemically or
heat-killed)
[0135] Representative antigens that can be used according to the
present invention include, but are not limited to, natural,
recombinant or synthetic products selected from viruses, bacteria,
fungi, parasites and other infectious agents e.g. prions. Antigens
may for example be an infectious agent selected from the following
infectious agents: Entamoeba histolytica, Bacillus including
Bacillus cereus and Bacillus subtilis group, Blastocystis hominis,
Bovine Spongiform Encephalopathy (BSE) and Creutzfeldt-Jakob
Disease (CJD) typical and atypical strains, Campylobacter including
Campylobacter jejuni, Vibrios including Vibrio cholerae,
Clostridium botulinum, Clostridium difficile, Clostridium
perfringens, Cryptosporidium, Cyclospora cayetanensis, Escherichia
coli, EnteroHemorrhagic Escherichia Coli (EHEC), Enterotoxigenic
Escherichia Coli (ETEC), Helicobacter pylori, Listeria
monocytogenes, Trichinella spiralis, Cryptosporidium including
Cryptosporidium parvum, Cyclospora cayetanensis, Enteroviruses,
Escherichia coli--including vero cytotoxin-producing (VTEC) strains
and others, Giardia including Giardia duodenalis, Giardia lamblia,
Giardia intestinalis, Hepatitis A virus, Listeria monocytogenes,
Marine biotoxins, (Norwalk-like viruses (NLV), small round
structured viruses (SRSV)), Rotavirus, Adenoviruses, Sapoviruses,
Astroviruses, Polio virus, Salmonella including Salmonella enterica
serovar Enteritidis, Salmonella typhimurium, Salmonella typhi and
Salmonella paratyphi, Shigella including Shigella sonnei, Shigella
boydii, Shigella dysenteriae and Shigella flexneri, Staphylococcus
aureus, Worms, helminthes, Yersinia for example Yersinia
enterocolitica and Yersinia pseudotuberculosis, or a combination
thereof.
[0136] Fungal antigens may for example be Candida albicans,
Aspergillus niger, Aspergillus fumigatus, Cryptococcus neoformans,
Pneumocystis carinii, Coccidioides posadasii, Pythium insidiosum,
or a combination thereof.
[0137] The compositions may comprise in combination a fungus and a
bacterium, and/or two or more strains of the same or different
bacterial species. The invention is intended to cover naturally
occurring strains as well as recombinant or synthetic microorganism
strains which for example express or over express antigenic
proteins.
[0138] In preferred embodiments the composition comprises ETEC as
active ingredient. It will be recalled that such compositions may
comprise an adjuvant, for example a glycolipid adjuvant such as,
for example, .alpha.-GalCer or an analogue thereof.
[0139] Also to be mentioned are compositions of the invention which
comprise Helicobacter pylori as active ingredient. It will be
recalled that such compositions may comprise an adjuvant, for
example a glycolipid adjuvant such as, for example, .alpha.-GalCer
or an analogue thereof.
[0140] Another composition of the invention comprises Vibrio
Cholerae (for example O1 V. Cholerae strains including the Ogawa
and Inaba serotypes and cells comprising antigens of both Ogawa and
Inaba serotypes, for example the cells described in WO2011/034495)
as active ingredient. A particular V. Cholerae strain is V Cholerae
JS1569, a rifampicin-resistant derivative of strain CVD103: J. B.
Kaper, H. Lockman, M. M. Baldini, M. M. Levine, Biotechnology, 2
(1984), pp. 345-349 and also V Cholerae JS1569 strains which have
been engineered to overexpress the Inaba LPS polypeptide. Such
compositions may comprise an adjuvant, for example a glycolipid
adjuvant such as, for example, .alpha.-GalCer or an analogue
thereof.
[0141] The invention therefore includes within its scope a
composition comprising:
a matrix comprising a hydrogel-forming polymer; and comprised in
the matrix, (i) a live, killed, attenuated or inactivated bacterium
that is selected from enterotoxigenic Escherichia Coli (ETEC),
Helicobacter pylori and V. Cholerae, (ii) a surfactant and (iii) an
adjuvant. For example in this embodiment the live, killed,
attenuated or inactivated bacterium that is selected from
enterotoxigenic Escherichia Coli (ETEC) and Helicobacter
pylori.
[0142] The invention therefore additionally includes within its
scope a composition comprising [0143] a surfactant, [0144] a live,
killed, attenuated or inactivated bacterium that is selected from
enterotoxigenic Escherichia Coli (ETEC), Helicobacter pylori and V.
Cholerae, for example selected from enterotoxigenic Escherichia
Coli (ETEC) and Helicobacter pylori, [0145] an adjuvant, and [0146]
a hydrogel-forming polymer in which the surfactant, the
microorganism and the adjuvant are included; and wherein the
composition when combined with water is capable of releasing
self-assembly structures comprising surfactant and adjuvant.
[0147] It will be understood by the reader that the entirety of the
disclosure of this specification is applicable to the compositions
of the preceding two paragraphs. A composition of either of the two
preceding paragraphs in which the bacterium is ETEC may therefore
include any one or more features described elsewhere herein as
optional (or preferred) features of the invention. A composition of
either of the two preceding paragraphs in which the bacterium is H.
pylori may therefore include any one or more features described
elsewhere herein as optional (or preferred) features of the
invention. Similarly a composition of either of the two preceding
paragraphs in which the bacterium is a V. Cholerae may therefore
include any one or more features described elsewhere herein as
optional (or preferred) features of the invention.
[0148] The invention includes embodiments in which the composition
comprises an active ingredient which is an adjuvant selected from
the group consisting of: immunostimulant; T-cell activator;
macrophage activator; saponins, fractions of saponins, synthesized
components of saponins, ISCOMS, muramyl dipeptide and analogues,
pluronic polyols, trehalose dimycolate, amine containing compounds,
cytokines, lipopolysaccharide derivatives and cationic transfection
reagents (e.g. DOTAP). Adjuvants may be chosen for example from the
ceramides (e.g. .alpha.-galactosylceramide also known as
.alpha.-GalCer), chitosan, cholera toxin e.g. rCTB (recombinant B
subunit of cholera toxin), E. coli heat labile enterotoxin e.g.
mLT, oligo-nucleotides e.g. oligodeoxynucleotides such as CpG
(cytosine phosphate guanine) and ODN1a
(deoxy-inosine/deoxy-cytosine) whether or not derivatised,
monophospholipid (MPL) e.g. MPLA, BCG, saponins including those
derived from the soap bark tree (Quillaja saponaria) such as QS21
and QuilA, Poly I:C (polyinosinic:polycytidylic acid or
polyinosinic-polycytidylic acid sodium salt), etc, various oils
such as, for example, cholesterol-related or cholesterol-derived
oils such as, for example, squalene (IUPAC name:
(6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexa-
eneoils. Derivatives of all the preceding substances are also
included whether or not derivatives are mentioned in a specific
context. Substances identified here as adjuvants may have or play
other roles in the invention or may play more than one role
simultaneously. For example, rCTB may also, in certain embodiments,
play the role of an antigen.
[0149] As adjuvants may also be mentioned marine derivatives,
sponges etc and their derivatives. In general, toll-like receptor
ligands may be included as adjuvants and include LPS, lipoproteins,
lipopeptides, flagelin, double-stranded RNA, unmethylated CpG
islands and various other forms of DNA and RNA classically released
by bacteria and viruses. TLR3 and TLR9 ligands are preferred in one
embodiment. Substances which bind to the CD1d protein on
antigen-presenting cells are particularly contemplated as are
mistletoe extracts, particularly detoxified mistletoe extracts.
Other adjuvants contemplated include the Nod-like receptor (NLR)
ligands described by Wagner et al in PLoS ONE, April 2009, Vol 4,
Issue 4, the entirety of which is incorporated herein by reference.
Muramyl dipeptide is also envisaged as is KLKL5KLK described by Li
et al in DNA and Cell Biology, Vol 27, No 8, 2008 the entirety of
which is incorporated herein by reference. Also contemplated is
KLKL5KLK in combination with ODN1a as described by Schellack et al
in Vaccine 24 (2006) 5461-5472, the entirety of which is
incorporate herein by reference.
[0150] Other adjuvants which may be included in the invention
include: amorphous aluminium hydroxyphosphate sulfate, aluminium
hydroxide, aluminium phosphate, aluminium potassium sulfate and
other aluminium compounds, Al hydrogel, cationic liposome-DNA
complex JVRS-1001SCOM(s).TM., calcium phosphate, Freund's Complete
Adjuvant, Freund's Incomplete Adjuvant, CpG DNA, cholera toxin B
subunit, liposomes, dimethyldioctadecylammonium bromide,
Escherichia coli non-toxic B subunit, IL-12, IL-15,
interleukin-1.beta., interleukin-2, interleukin-7, Escherichia coli
heat-labile toxin LTK63, LTK72, TiterMax Gold, Ribi Adjuvant System
(RAS), Montanide ISA 720, Montanide Incomplete Seppic,
Corynebacterium-derived P40, MPL.TM., alum and lipopolysaccharide
(LPS) derivative Monophosphoryl Lipid A (MPL) combination (AS04),
MF59 oil-in-water emulsion with MPL and saponin fraction QS21
(AS02), AS03, Bacterial lipopolysaccharide (LPS), muramyl dipeptide
(MDP), CRL1005 copolymer, killed Corynebacterium parvum, Montanide
ISA 51, Bordetella pertussis, cationic liposomes, adamantylamide
dipeptide (AdDP), Arlacel A, VSA-3, POLYGEN.TM., Adjumer.TM., Algal
Glucan,
N-(2-deoxy-2-L-leucylamino-.beta.-D-glucopyranosyl)-N-octadecyldodecanoyl-
amide hydroacetate,
N-acetylglucosaminyl-N-acetylinuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxy
propylamide (DTP-DPP), stearyl tyrosine, Specol, linear
(unbranched) .beta.-D-(2-1) polyfructofuranosyl-.alpha.-D-glucose
and Al hydrogel (Algammulin),
N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl) propanediamine, Calcium
Phosphate Gel, CTA1-DD gene fusion protein, DOC/Al(OH)3/mineral
carrier complex, .gamma.-Inulin, Gerbu Adjuvant,
granulocyte-macrophage colony stimulating factor,
N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine,
recombinant hIFN-gamma/Interferon-g, Sclavo peptide, Rehydragel LV,
Rehydragel HPA, Loxoribine, MF59, MTP-PE liposomes, murametide,
murapalmitine, D-murapalmitine, neuraminidase-galactose oxidase,
non-ionic surfactant vesicles (NISV), polymethyl methacrylate,
protein cochleates, Stimulon.TM. QS-21, SPT (Antigen Formulation),
Quit-A, 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl
2-deoxy-4-O-phosphono-3-O--[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3--
tetradecanoyoxytetradecanoylamino]-.beta.-D-glucopyranos
idetriethylammonium salt (RC529), LTR192G, E. coli heat-labile
toxin, 1-(2-methypropyl)-IH-imidazo[4,5-c]quinolin-4-amine
(Imiquimod), Resiquimod, AF03, Flagellin protein, ISCOMATRIX.RTM.,
Abisco-100, albumin-heparin microparticles, B7-2 (CD86),
dehydroepiandrosterone, immunoliposomes containing antibodies to
costimulatory molecules, SAF-1, Sendai proteoliposomes, threonyl
muramyl dipeptide (TMDP), Ty-VLPs, Bupivacaine, polyester poly
(DL-lactide-co-glycolide), monophosphoryl lipid A (MPL)+squalene,
non-toxic mutant E112K of cholera toxin mCT-E112K, Matrix-S.
[0151] Preferred adjuvants include the ceramides and other lipid
molecules (especially non-ionic lipid molecules) which specifically
stimulate natural killer T (NKT) cells. A ceramide is composed of
sphingosine and a fatty acid and are found in high concentrations
within the cell membrane of cells being one of the component lipids
that make up sphingomyelin, one of the major lipids in the lipid
bilayer. Ceramide can act as a signaling molecule e.g. regulating
the differentiation, proliferation, programmed cell death (PCD),
and apoptosis (Type I PCD) of cells. Preferred ceramides include
alpha-galactosylceramides including agelasphins and derivatives. A
particularly preferred alpha-galactosylceramide is the product
known as KRN7000 commercially available from Funakoshi, Japan, and
originally synthesised by Kirin Pharmaceuticals, Japan. Derivatives
of KRN7000 are also contemplated as components of the composition
of the invention and are described in detail by WO 2004/028475 and
Dere et al (2008) in Organic Letters, Vol 10, n.degree. 20, pp
4641-4644, the entirety of both of which are incorporated herein by
reference. The thiolated derivative of alpha-galactosylceramide (in
which the glycosidic oxygen atom has been replaced by a sulphur
atom) described by Dere et al is particularly preferred as are
racemates, enantiomers or diastereoisomers thereof and of closely
related derivatives.
[0152] Ceramide derivatives including analogues of .alpha.-GalCer
which may be used in the invention as adjuvants include
1,2,3-triazole containing analogues of .alpha.-GalCer;
3-O-sulfo-.alpha.-GalCer; 4'-O-sulfo-.alpha.-GalCer;
6'-O-sulfo-.alpha.-GalCer; 4-deoxy and 3,4-dideoxy analogues of
.alpha.-GalCer; .alpha.-GalCer analogues containing an aromatic
ring; C-glycoside analogues of .alpha.-GalCer; disaccharide
analogues of .alpha.-GalCer including
Gal.alpha.1-6Gal.alpha.1-1'Cer, Gal.alpha.1-2Gal.alpha.1-1'Cer,
Gal.alpha.1-4Glc.beta.1-1'Cer, Gal.alpha.1-6Glc.alpha.1-1'Cer,
.beta.1-3Gal.alpha.1-1'Cer; galactosyl serine-type ceramide;
GalA-GSL; Gal.alpha.1-2GalCer, Gal.alpha.1-3GalCer,
Gal.alpha.1-6GalCer glycolipids; glycoside analogues of
.alpha.-GalCer; pegylated derivatives of .alpha.-galactosylceramide
(aGCPEG); Sphingomonas glycosphingolipids; .alpha.-anomeric GalCer;
C-glycoside analogues of .alpha.-GalCer including .alpha.-C-GalCer;
.alpha. mannosyl ceramide; .alpha.-galactosyl 1,2-diacyl
sn-glycerol; .alpha.-Galactosylceramide Analogue 8;
.alpha.-galacturonosyl Ceramide (Gal A-GSL); .alpha.-GalCer
analogues with one or more double bonds in the fatty acyl chain
including the C20:1 cis/trans C20:2 .alpha.-GalCer analogues;
.alpha.-GalCer analogues bearing a 6-phenylhexanoyl (C6Ph),
8-phenyloctanoyl (C8Ph), or 11-phenylundecanoyl group (C11Ph) as
the fatty acyl chain; .alpha.-glucosylceramide (.alpha.-GlcCer);
.alpha.-glucuronosyl ceramide; .alpha.-linked mannosyl ceramide
(.alpha.-ManCer); .beta.-anomeric GalCer; .beta.-GalCer;
.beta.-GlcCer; ceramide dihexosyl sulfate; ceramide trihexosyl
sulfate; ceramide tetrahexosyl sulfate; isoglobotrihexosylceramide;
OCH (analogue of .alpha.-GalCer with a truncated sphingosine tail);
or PBS-25 (analogue of .alpha.-GalCer having an eight carbonyl acyl
chain).
[0153] Further examples of lipid adjuvants which may be used in the
invention include Borelia glycolipids; glycosphingolipid (GSL);
Phospholipids including diphosphatidylglycerol,
phosphatidylethanolamine, phosphatidylcholine,
phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,
lyso-PC, lyso-phosphatidylethanolamine, lyso-phophatidylserine and
lyso-phosphatidylinositol; CD1 lipids including isoforms CD1a,
CD1b, CD1C, CD1d.
[0154] In one embodiment of the invention the adjuvant is not
.alpha.-GalCer.
[0155] In one embodiment, the inclusion in the composition of the
invention of more than one adjuvant may aid in the stimulation of a
mucosal immune response.
[0156] In embodiments the adjuvant is present in the composition at
0.01% to 1%, for example 0.05 to 0.5% or 0.1 to 0.3%, wherein the %
is by weight based upon the dry weight of the composition.
[0157] In preferred embodiments the active ingredient is a
microorganism and a second active ingredient is an adjuvant.
Preferably the adjuvant is an immunostimulant e.g. is a T-cell
activator and/or an antigen-presenting cell activator. A
representative example of an adjuvant is .alpha.-GalCer.
[0158] In certain embodiments the weight ratio of
adjuvant:microorganism is from 1:1 to 1:10 and optionally from 1:3
to 1:5, e.g. from 1:1.35 to 1:1.45. The microorganism may consist
of one or more unicellular microorganisms. In this embodiment, the
ratio of adjuvant to the aggregate amount of unicellular
microorganisms (mg dry weight of adjuvant to 10 10 cells) may be
from 0.1-100 mg:10 10 cells, for example 0.1-10 mg:10 10 cells,
e.g. 0.25-5 mg:10 10 cells, particularly 0.4-5 mg:10 10 cells.
[0159] In certain embodiments, wherein the microorganism consists
of one or more unicellular microorganisms, the ratio of surfactant
to the aggregate amount of unicellular microorganisms (mg dry
weight of surfactant to 10 10 cells) may be from 10-200 mg:10 10
cells and optionally from 25-125 mg:10 10 cells, e.g. from 25-150
mg:10 10 cells, 25-100 mg:10 10 cells, 50-200 mg:10 10 cells,
50-100 mg:10 10 cells or 60-90 mg:10 10 cells.
[0160] In embodiments where any component of the composition are
temperature sensitive (e.g. a microorganism, an adjuvant etc.) it
is appreciated that methods of manufacture that accommodate
temperature labile components (as described below) may be used.
[0161] The composition of the invention may be utilized in order to
bring antigen into contact with the gut-associated lymphoid tissue
(GALT) either directly or after absorption. The composition of the
invention is intended, in one embodiment, to allow antigens and/or
adjuvants to interact with or facilitate their interaction with T
cells in the GALT. In one embodiment, the section of the GI tract
where this interaction occurs is the rectum and/or colon. In
another embodiment, the section is the jejunum or other site having
almost no immune inductive sites. The composition of the invention
may be adapted to release the microorganism and adjuvant after the
composition has passed through the stomach and particularly in the
upper small intestine; the composition may therefore be enteric
coated.
[0162] The present invention provides compositions and/or
formulations comprising the necessary antigenic peptides (including
any covalently or non-covalently modified peptides) to be
formulated, with or without adjuvants and optionally other
ingredients as described elsewhere herein. Such other ingredients
e.g. permeability enhancers, along with the composition of the
invention being optionally encapsulated (e.g. coated) with a single
or multiple layer(s) of (for example) a polymer, with the layers or
polymer coatings being modified permit release of the active
components at the most appropriate location along the intestine or
colon/rectum.
[0163] Accordingly, the invention includes a method of inducing an
immune response in a mammal, e.g. a human, comprising:
[0164] administering to the mammal a composition of the disclosure
which is adapted to release a microorganism selected from live,
killed, attenuated and inactivated microorganisms in the GI tract;
and/or
[0165] administering to the mammal a composition of the disclosure
which further comprises an adjuvant.
[0166] When used to induce an immune response, for example as a
vaccine or as an immunotherapeutic agent the dose of the
microorganism and vaccine will naturally need to be varied
depending upon the specific microorganism used and the nature of
the use of the composition. Suitably the composition provides a
dose of 10.sup.8 to 10.sup.14 cells. The size of the dose required
may be determined by a skilled person using well known methods. The
dose may be administered as a single bolus dose or as a divided
dose. Dosage may be repeated as required to provide the desired
immune response, For example repeating administration of a
composition according to the invention may be carried out less than
a week after the initial dose. Alternatively administration may be
repeated after a period of at least one week. Optionally further
dosages can be given, each administration being separated from each
other, for example where each administration is separated by at
least one week. When repeat dosages are administered each dose may
be the same or different to the initial dose.
[0167] As adaptations for release in the colon or rectum may be
mentioned by way of example: [0168] formulating the composition as
a suppository [0169] formulating the composition for oral
administration and including release-controlling agents.
[0170] As examples of release-controlling agents, the composition
may comprise a polymer which is degraded by bacterial enzymes in
the colon or which otherwise acts as a barrier until the
composition reaches the colon (e.g. which is dissolved or degraded
in the conditions of the colon). Retardant polymers which are
degraded or eroded during passage down the GI tract may be used
and/or pH-independent polymers comprising pore-formers which are
dissolved or degraded in the conditions of the colon. The
composition may include an enteric polymer to prevent degradation
in the stomach such that the composition is exposed to further
dissolution, erosion or degradation only when it has entered the
intestine. Polymers mentioned in this paragraph may be included in
the matrix and/or may form or be comprised in one or more
coatings.
[0171] As discussed elsewhere herein, pH-independent coating
polymers may be used, for example ethylcellulose. The addition,
therefore, to an ethylcellulose (e.g. Surelease.TM.) or other
pH-independent coating of a second polymer (e.g. a polysaccharide,
especially a heteropolysaccharide) which is susceptible to
degradation by bacterial enzymes but not by digestive enzymes, e.g.
human digestive enzymes, helps ensure that the barrier function of
the coating is destroyed by the action of such enzymes in the
terminal ileum and/or colon, thereby ensuring release of the
actives in the ileum and/or colon. The inclusion of such a
bacterial enzyme-degradable polymer in a pH-independent coating,
e.g. ethylcellulose, provides flexibility in modulating the amount
of polymer added to the beads of the invention in order to achieve
optimal dissolution profiles. In general terms, therefore, the
disclosure includes formulations as described herein which comprise
a coating comprising a combination of a delayed release material,
for example an erodible polymer e.g. ethylcellulose, and a polymer
susceptible of degradation by bacterial enzymes in the colon, e.g.
a polysaccharide and particularly a water-soluble polysaccharide,
particularly a pectin. However, even in the case of targeted
colonic release, it is not mandatory to combine ethylcellulose or
other pH-independent coating polymer with a polymer susceptible to
degradation by bacterial enzymes.
Nutrients
[0172] The compositions of the invention may further comprise an
immune-enhancing nutrient, for example one or more nutrients
selected from vitamins A, B (e.g. one or a combination of vitamin
B6, vitamin B12, niacin (vitamin B3), pantothenic acid, riboflavin
(vitamin B2), thiamin (vitamin B1) and folic acid), vitamin C,
vitamin E; carotenoids, e.g. beta-carotene, iron, manganese,
selenium and zinc. The composition may comprise a nutrient, e.g. a
combination of nutrients, in the matrix (included in the
hydrogel-forming polymer); the composition may comprise a nutrient,
e.g. a combination of nutrients, in association with the
surfactant; the composition may comprise a nutrient, e.g. a
combination of nutrients in a coating; the composition may comprise
a nutrient (e.g. a combination of nutrients) in each of two or
three of the aforesaid locations. Water soluble nutrients may be
suitable for inclusion in the matrix (dried aqueous phase) and
surfactant-soluble nutrients may be suitable for association with,
e.g. inclusion in, the surfactant, but the invention is not limited
to these possibilities.
Polymer Matrix
[0173] The disclosure includes formulations comprising a surfactant
phase and a continuous phase or matrix phase to provide mechanical
strength. The continuous phase or matrix phase comprises a
hydrogel-forming polymer. Such formulations therefore comprise a
polymer matrix.
[0174] A hydrogel-forming polymer is a polymer capable of forming a
hydrogel. A hydrogel may be described as a solid or semi-solid
material, which exhibits no flow when at rest, comprising a network
(matrix) of hydrophilic polymer chains that span the volume of an
aqueous liquid medium.
[0175] The composition may comprise a hydrogel-forming polymer
selected from the group consisting of: gelatin; agar; agarose;
pectin; carrageenan; chitosan; alginate; starch; xanthan gum; gum
Arabic; guar gum; locust bean gum; polyurethane; polyether
polyurethane; cellulose; cellulose ester, cellulose acetate,
cellulose triacetate; cross-bonded polyvinyl alcohol; polymers and
copolymers of acrylic acid, hydroxyalkyl acrylates, hydroxyethyl
acrylate, diethylene glycol monoacrylate, 2-hydroxypropylacrylate,
3-hydroxypropyl acrylate; polymers and copolymers of methacrylic
acid, hydroxyethyl methacrylate, diethyleneglycol monomethacrylate,
2-hydroxypropyl methacrylate, 3-hydroxypropyl methacrylate,
dipropylene glycol monomethylacrylate; vinylpyrrolidone; acrylamide
polymers and copolymers, N-methylacrylamide, N-propylacrylamide;
methacrylamide polymers and copolymers, N-isopropylmethacrylamide,
N-2-hydroxyethylmethacrylamide; and vinyl pyrrolidone; and
combinations thereof. In specific embodiments binary or tertiary
etc combinations of any of the above substances are foreseen.
[0176] The hydrogel-forming polymer may also be referred to as a
hydrocolloid i.e. a colloid system wherein the colloid particles
are dispersed in water and the quantity of water available allows
for the formation of a gel. In embodiments it is preferred to use
reversible hydrocolloids preferably thermo-reversible hydrocolloids
(e.g. agar, agarose, gelatin etc) as opposed to irreversible
(single-state) hydrocolloids. Thermo-reversible hydrocolloids can
exist in a gel and sol state, and alternate between states with the
addition or elimination of heat. Gelatin, agar and agarose are
thermo-reversible, rehydratable colloids and are particularly
preferred. Gelatin derivatives such as, for example, succinated or
phthalated gelatins are also contemplated. Thermoreversible
hydrocolloids which may be used according to the invention include
those derived from natural sources such as, for example,
carrageenan (extracted from seaweed), gelatin (extracted from
bovine, porcine, fish or vegetal sources), agar (from seaweed),
agarose (a polysaccharide obtained from agar) and pectin (extracted
from citrus peel, apple and other fruits). A non-animal based
hydrocolloid may be preferred for certain applications e.g.
administration to vegetarians or to individuals not wishing to
ingest animal products for religious or health reasons. In relation
to the use of carrageenan, reference is made to US patent
application 2006/0029660 A1 (Fonkwe et al), the entirety of which
is incorporated herein by reference. The hydrogel-forming polymer
may comprise or be a combination of gelatin with one or more other
thermoreversible hydrocolloids, e.g. with one or more other of the
thermoreversible hydrocolloids just listed. The hydrogel-forming
polymer may comprise or be a combination of gelatin with agar;
optionally, at least one further thermoreversible hydrocolloid may
be included in the combination, for example one just listed.
[0177] Thermo-reversible colloids present a benefit over other
hydrogel-forming polymers. Gelation or hardening of
thermo-reversible colloids occurs by cooling the colloid, e.g. in a
liquid cooling bath or by air flow. Gelation of other
hydrogel-forming polymers, which is chemically driven, can lead to
leakage of the composition contents into the gelation medium as the
hardening process can take time to occur. Leakage of the content of
the composition may lead to an inaccurate quantity of the active
ingredient within the composition. Thermo-reversible colloids are
also known as thermo-reversible gels, and it is therefore preferred
that the hydrogel former be a thermo-reversible gelling agent.
[0178] Another term which may be applied to hydrogel formers which
are advantageous is "thermotropic": a thermotropic gelling agent
(which the reader will infer is preferred as a hydrogel former used
in the invention) is one caused to gel by a change in temperature
and such gelling agents are able to gel more rapidly than those
whose gelling is chemically induced, e.g. ionotropic gelling agents
whose gelling is induced by ions, for example chitosan. In
embodiments of the invention, therefore, the hydrogel former is a
thermotropic gel-forming polymer or a combination of such
polymers.
[0179] The manufacture of the composition may require that the
hydrogel-forming polymer be present as a solution, which is
preferably an aqueous solution. The hydrogel-forming polymer
represents between 5% and 50%, preferably between 10% and 30%,
still more preferably between 15% and 20% by weight of the aqueous
phase during manufacture.
[0180] In embodiments the composition comprises at least 25%,
suitably at least 40% by weight based upon the dry weight of the
composition of the hydrogel-forming polymer. For example the
hydrogel-forming polymer is present form 25 to 70%, for example 40
to 70% suitably 45 to 60% of the composition, wherein the % is by
weight based upon the dry weight of the composition.
[0181] In embodiments the hydrogel-forming polymer is a
pharmaceutically acceptable polymer.
[0182] In certain embodiments the hydrogel-forming polymer is
gelatin. In certain embodiments the hydrogel-forming polymer
comprises gelatin. In certain embodiments the gelatin comprises at
least 40%, for example 40 to 70% suitably 45 to 60% of the
composition, wherein the % is by weight based upon the dry weight
of the composition.
[0183] The hydrogel-forming polymer may optionally comprise a
plasticiser for example sorbitol or glycerine, or a combination
thereof. In particular one or more plasticisers may be combined
with gelatin.
[0184] In embodiments in which gelatin is the polymer matrix of the
invention, reference is hereby made to "Bloom strength", a measure
of the strength of a gel or gelatin developed in 1925 by O. T.
Bloom. The test determines the weight (in grams) needed by a probe
(normally with a diameter of 0.5 inch) to deflect the surface of
the gel 4 mm without breaking it. The result is expressed in Bloom
(grades) and usually ranges between 30 and 300 Bloom. To perform
the Bloom test on gelatin, a 6.67% gelatin solution is kept for
17-18 hours at 10.degree. C. prior to being tested.
[0185] When the hydrogel-forming polymer comprises or is gelatin
the bloom strength of the gelatin may be in the range of 125 Bloom
to 300 Bloom, 200 Bloom to 300 Bloom and preferably 250 Bloom to
300 Bloom. It should be appreciated that higher bloom strength
gelatin can be replaced by lower bloom strength gelatin at higher
concentrations.
[0186] According to the invention, in embodiments in which the
water-soluble polymer matrix material comprises or is gelatin, the
gelatin may be sourced by a variety of means. For example, it can
be obtained by the partial hydrolysis of collagenous material, such
as the skin, white connective tissues, or bones of animals. Type A
gelatin is derived mainly from porcine skins by acid processing,
and exhibits an isoelectric point between pH 7 and pH 9, while Type
B gelatin is derived from alkaline processing of bones and animal
(bovine) skins and exhibits an isoelectric point between pH 4.7 and
pH 5.2. Type A gelatin is somewhat preferred. Gelatin for use in
the invention may also be derived from the skin of cold water fish.
Blends of Type A and Type B gelatins can be used in the invention
to obtain a gelatin with the requisite viscosity and bloom strength
characteristics for bead manufacture.
[0187] Lower temperature gelatin (or gelatin derivatives or
mixtures of gelatins with melting point reducers) or other polymer
matrices able to be solidified at lower temperatures (e.g. sodium
alginate) are preferred for example when the active principle to be
incorporated in the composition of the invention is
temperature-labile or whose activity may be affected by exposure to
higher temperatures. It is therefore believed that polymer which
comprises or is low temperature gelatin is a preferred matrix
polymer in this invention.
[0188] According to the invention, in embodiments in which the
polymer comprises or is gelatin, the starting gelatin material is
preferably modified before manufacture to produce "soft gelatin" by
the addition of a plasticizer or softener to the gelatin to adjust
the hardness of the composition of the invention. The addition of
plasticizer achieves enhanced softness and flexibility as may be
desirable to optimise dissolution and/or further processing such
as, for example, coating. Useful plasticizers of the present
invention for combination with gelatin or another hydrogel-forming
polymer include glycerine (1,2,3-propanetriol), D-sorbitol
(D-glucitol), sorbitol BP (a non-crystallizing sorbitol solution)
or an aqueous solution of D-sorbitol, sorbitans (e.g. Andidriborb
85/70), mannitol, maltitol, gum arabic, triethyl citrate,
tri-n-butyl citrate, dibutylsebacate. Other or similar low
molecular weight polyols are also contemplated for example ethylene
glycol and propylene glycol. Polyethylene glycol and polypropylene
glycol may also be used although these are less preferred.
Glycerine and D-sorbitol may be obtained from the Sigma Chemical
Company, St. Louis, Mo. USA or Roquette, France. Some active agents
and excipients included for other functions may act as
plasticisers.
[0189] Softeners or plasticisers, if utilized, can be ideally
incorporated in a proportion rising to 30%, preferably up to 20%
and more preferably up to 10% by dry weight of the composition of
the invention, even more preferably between 3 and 8%, and most
preferably between 4% and 6%.
[0190] It is possible within the scope of the invention that the
hydrogel-forming polymer comprises a further surfactant, in
addition to said surfactant with which any adjuvant is associated,
which it will be recalled may be a mixture of surfactant
compounds.
[0191] Although not essential, the hydrogel-forming polymer may
also optionally contain a disintegrant where it is particularly
desired to enhance the rate of disintegration of the composition of
the invention. Examples of disintegrants which may be included are
alginic acid, croscarmellose sodium, crospovidone, low-substituted
hydroxypropyl cellulose and sodium starch glycolate.
[0192] A crystallisation inhibitor (e.g. approximately 1% by dry
weight of the composition) may also be included in the composition
of the invention. An example is hydroxy propyl/methyl cellulose
(HMC or HPMC, hypromellose etc) which may play other roles such as,
for example, emulsifier.
[0193] In an alternative preferred embodiment, the polymer matrix
is chitosan which can exist in the form of biogels with or without
additives as described e.g. in U.S. Pat. No. 4,659,700 (Johnson
& Johnson); by Kumar Majeti N. V. Ravi in Reactive and
Functional Polymers, 46, 1, 2000; and by Paul et al. in ST.P.
Pharma Science, 10, 5, 2000 the entirety of all 3 of which is
incorporated herein by reference. Chitosan derivatives e.g.
thiolated entities are also contemplated.
[0194] The hydrogel-forming polymer may have a low water content,
therefore the composition may have a low water content.
[0195] In certain embodiments the composition does not comprise
compounds containing a disulphide bond. In embodiments the
hydrogel-forming polymer does not comprise compounds containing a
disulphide bond.
Surfactant
[0196] The surfactant may be present as self-assembly structures
(e.g. micelles) dispersed within the hydrogel-forming polymer in a
"wet" (not yet dried) composition made as an intermediate in the
manufacturing process described herein. It is believed also to be
present as self-assembly structures (e.g. micelles) in the dried
composition but observability of self-assembly structures like
micelles or micelle-like structures in the dried composition is not
a requirement of the invention. It is mentioned at this point that
the presence of a surfactant in a self-assembly structure (e.g.
micelle) form does not require that the entire surfactant content
of a composition is in this form as it is considered more probable
that a portion of the surfactant will be outside the self-assembly
structures (e.g. micelles). Thus in the "wet" composition, whether
the hydrogel-forming polymer is in the gel state or the sol
(liquid) state may comprise the surfactant at a concentration above
the critical concentration for formation of self-assembly
structures (e.g. micelles) (i.e. above the critical micelle
concentration).
[0197] With regard to micelles, the diameter of the dispersed
micelles is between 0.5 nm and 200 nm, 1 nm and 50 nm, or 5 nm and
25 nm. The size of the micelles may be determined by dynamic light
scattering or diffusion NMR techniques known within the art.
Although the size of the micelles is given as a diameter this does
not imply that the micelles must be purely spherical species only
that they may possess some approximately circular dimension.
[0198] The surfactant may be a non-ionic surfactant. The surfactant
may be a polyoxyethylated surfactant. The surfactant has a
hydrophilic head which may be a hydrophilic chain, for example a
polyoxyethylene chain or a polyhydroxylated chain.
[0199] The surfactant of course has a hydrophobic part and in
particular a hydrophobic chain. The hydrophobic chain may be a
hydrocarbon chain, for example having at least 6 carbon atoms and
optionally at least 10 carbon atoms, and particularly of at least
12 carbon atoms; some hydrocarbon chains have no more than 22
carbon atoms, for example C.sub.10-C.sub.20, C.sub.12-C.sub.20 or
C.sub.15-C.sub.20 hydrocarbon chains. It may be an alkyl chain,
e.g. having a number of carbon atoms just mentioned. It may be an
alkenyl chain comprising one or more carbon-carbon double bonds,
e.g. having a number of carbon atoms just mentioned. The surfactant
may comprise a hydrocarbon chain, e.g. alkyl chain or alkenyl
chain, that is substituted provided that it maintains a hydrophobic
characteristic. There may for example be one or two substituents,
for example a single substituent, e.g. selected from halogen (e.g.
F or Cl), hydroxy, thiol, oxo, nitro, cyano; hydroxy or thiol
substituents may be esterified by for example a fatty acid. One
class of surfactants comprise a hydrocarbyl moiety monosubstituted
by hydroxy; optionally, at least a portion of the hydroxy groups of
an aliquot of surfactant, e.g. of the surfactant in a bead, may be
esterified by a fatty acid or mono-hydroxy fatty acid as disclosed
herein or etherified by a fatty alcohol for example having at least
6 carbon atoms and optionally at least 10 carbon atoms, and
particularly of at least 12 carbon atoms; some hydrocarbon chains
have no more than 22 carbon atoms, for example C.sub.10-C.sub.20,
C.sub.12-C.sub.20 or C.sub.15-C.sub.20 fatty alcohols.
[0200] The hydrophobic chain may be part of an esterified fatty
acid R.sup.1--COOH or of an etherified or esterified fatty alcohol
R.sup.1--COH where R.sup.1 is the hydrophobic chain, e.g. as
mentioned in the preceding paragraph. The ester-forming or, as the
case may be, ether-forming group will typically comprise a
hydrophilic chain. A portion of the fatty acid molecules
R.sup.1--COOH or fatty alcohol molecules R.sup.1--COH may be as the
free acid or alcohol and a portion may be esterified or, in the
case of fatty alcohols, etherified.
[0201] As mentioned, the surfactant may have a hydrophilic chain
and may be a non-ionic surfactant, and may satisfy both
requirements. The hydrophilic chain may be a poly(ethyleneglycol),
also known as poly(oxyethylene) or macrogol. The hydrophilic chain
may be of the formula --(O--CH.sub.2--CH.sub.2).sub.n--OR where n
is 5 or 6 to 50 and R is H or alkyl, e.g. ethyl or methyl. The
invention includes implementations in which n is from 6 to 40, e.g.
from 6 to 35. In some embodiments, n is from 6 to 25 and optionally
is from 8 to 25 or from 8 to 15. In other embodiments, n is from 8
to 50 or from 8 to 40, e.g. is from 10 to 50, 10 to 40 or 10 to 35.
In a particular embodiment, n is 15. For all hydrophilic chains of
the formula --(O--CH.sub.2--CH.sub.2).sub.n--OR, in one class of
embodiments R is H.
[0202] The hydrophilic chain may be a polyhydroxylated chain (for
example a C.sub.5-C.sub.20 e.g. C.sub.5-C.sub.10 chain), e.g.
having a hydroxy group on the carbon atoms of the chain, for
example a glucamide.
[0203] The surfactant may comprise a combination of a hydrophobic
chain as described above and a hydrophilic chain as described
above. It may therefore be, or comprise, a macrogol ester of a
fatty acid as described herein or a macrogol ether of a fatty
alcohol as described herein.
[0204] Micelle-forming surfactants comprising a hydrophobic chain
and a hydrophilic chain can be selected from the group consisting
of: macrogol esters; macrogol ethers; diblock copolymers; triblock
copolymers; and amphiphilic polymers. In certain embodiments of the
invention any combinations of the group are included within the
invention.
[0205] Examples of macrogol esters which are suitable for use in
the present invention are macrogol esters of fatty acids having at
least 6 carbon atoms and optionally at least 10 carbon atoms, and
particularly of at least 12 carbon atoms; some fatty acids have no
more than 22 carbon atoms, for example C.sub.10-C.sub.20,
C.sub.12-C.sub.20 or C.sub.15-C.sub.20 fatty acids. The fatty acids
may be saturated or unsaturated but are in particular saturated. To
be mentioned are macrogol 25 cetostearyl ether (Cremophor.RTM.
A25); macrogol 6 cetostearyl ether (Cremophor.RTM. A6); macrogol
glycerol ricinoleate 35 (Cremophor.RTM. EL); macrogol-glycerol
hydroxystearate 40 (Cremophor.RTM. RH 40);
macrogol-15-hydroxystearate (polyoxyl-15-hydroxystearate US
Pharmacopoeia and National Formulary, European Pharmacopoeia, e.g.
Kolliphor HS 15, previously known as Solutol.RTM. HS 15). Examples
of macrogol ethers which are suitable for use in the present
invention are macrogol ethers of fatty alcohols having at least 6
carbon atoms and optionally at least 10 carbon atoms, and
particularly of at least 12 carbon atoms; some fatty alcohols have
no more than 22 carbon atoms, for example C.sub.10-C.sub.20,
C.sub.12-C.sub.20 or C.sub.15-C.sub.20 fatty alcohols. The fatty
alcohols may be saturate or unsaturated but are in one embodiment
saturated. Kolliphor.RTM. HS 15 is obtained by reacting 15 moles of
ethylene oxide with 1 mole of 12-hydroxy stearic acid; the
surfactant may therefore be or comprise a surfactant obtainable by
(having the characteristics of a surfactant obtained by) reacting
10-25 moles of ethylene oxide with 1 mole of 12-hydroxy stearic
acid; the number of moles of ethylene oxide may, from 12-25 and
optionally from 15-20, e.g. 15 or 20.
[0206] Kolliphor.RTM. HS 15 consists of polyglycol mono- and
di-esters of 12-hydroxystearic acid and about 30% of free
polyethylene glycol. The main components of the ester part have the
following chemical structures:
##STR00001##
where x and y are integers and a small part of the 12-hydroxy group
can be etherified with polyethylene glycol.
[0207] Therefore, the surfactant may comprise a mixture of
molecules. For example, the surfactant composition used in the
manufacturing process may comprise a polyethoxylated (PEGylated)
molecule and comprise additionally free polyethoxy (free PEG)
compound, or the surfactant composition used in the manufacturing
process may comprise a molecule having a polyhydroxylated moiety
and comprise additionally free polyhydroxy compound. Amongst the
implementations of the invention are those in which the surfactant
is, or comprises, a PEGylated fatty acid, e.g. a PEGylated hydroxy
fatty acid, in combination with free PEG.
[0208] Examples of amphiphilic polymers which are suitable for use
in the present invention are: alkyl glucamides; fatty alcohol
poly(ethoxyl)ates also known as polyethoxylated alkyl ethers;
poly(ethoxyl)ated fatty acid esters (Myrj or Kolliphor); fatty
amide polyethoxylate; fatty amine ethoxylate; alkylphenol
ethoxylate; polyethoxylated sorbitan esters (polysorbates);
polyethoxylated glycerides; or poly-glycerol esters.
[0209] Examples of copolymers, which are suitable for use in the
present invention are: pluronics (poloxamers);
polyvinylpyrollidone-polyvinylacetate (Plasdone S630); aminoalkyl
methacrylate copolymer (Eudragit EPO); methacrylic acid-methyl
methacrylate copolymer (Eudragit S100, L100); polycaprolactone-PEG;
polycaprolactone-methoxy-PEG; poly(aspartic acid)-PEG;
poly(benzyl-L-glutamate)-PEG; poly(D,L-lactide)methoxy-PEG;
poly(benzyl-L-aspartate-PEG; or poly(L-lysine)-PEG
[0210] In a preferred embodiment the surfactant is a macrogol
ester, more preferably a macrogol ester that conforms to the
European Pharmacopoeia monograph number 2052
macrogol-15-hydroxystearate, such as Kolliphor.RTM. HS 15 marketed
by BASF.
[0211] Suitable surfactants comprise those which during manufacture
combine with the aqueous phase (including hydrogel-forming polymer)
in an amount above their CMC to form a clear liquid. Kolliphor.RTM.
HS 15 is such a surfactant.
[0212] In certain embodiments the weight ratio of the surfactant to
the microorganism antigen is from 10:1 to 100:1, optionally from
50:1 to 100:1. In some embodiments, the ratio is from 80:1 to 90:1.
In particular embodiments, the ratio is from 50:1 to 60:1.
[0213] In embodiments the surfactant is present in the composition
at 20 to 55%, for example 30 to 45% or 30 to 40% by weight, based
upon the dry weight of the composition.
Combinations of Self-Assembly Structure-Forming Compounds
[0214] In particular embodiments, the compositions of the invention
comprise a combination of self-assembly structure-forming
compounds. Such a combination of self-assembly structure-forming
compounds may consist of two or more surfactants as mentioned in
the preceding section of this specification. Alternatively, a
surfactant may be combined with one or more other compounds at
least potentially able to form micelles with the surfactant,
optionally selected from cationic lipids and glycolipids, amongst
others. As an additional option, a composition may comprise a
plurality of surfactants as mentioned in the preceding section of
this specification and one or more other compounds at least
potentially able to form self-assembly structures (e.g. micelles)
with the surfactant, optionally selected from cationic lipids and
glycolipids, amongst others.
[0215] In relation to "mixed self-assembly structures" or "mixed
micelles" comprising a combination of self-assembly
structure-forming (e.g. micelle-forming) compounds, it is believed,
but without being bound by theory, that at least a portion of
adjuvant .alpha.-GalCer will, together with said self-assembly
structure-forming surfactant, act as a self-assembly
structure-forming compound in these mixed structures. Other
glycolipids and ceramides will behave similarly, it is believed,
whether used singly or in combination with another glycolipid or
ceramide.
[0216] The invention therefore includes compositions as described
herein which comprise: [0217] two or more self-assembly
structure-forming (e.g. micelle-forming) surfactants, e.g. two or
more surfactants having a hydrophobic chain and a hydrophilic chain
[0218] a compound, e.g. a single compound or two or more compounds,
selected from cationic lipids and glycolipids [0219] two or more
self-assembly structure-forming (e.g. micelle-forming) surfactants
and a compound, e.g. a single compound or two or more compounds,
selected from cationic lipids and glycolipids.
Further Excipients
[0220] The invention foresees incorporation into the composition of
one or more of the following substances or categories of substances
in addition to the at least one active ingredient, the surfactant
and the hydrogel-forming polymer. For example, the composition may
contain a protectant such as, for example, a proteolytic enzyme
inhibitor or a protector against acid degradation or both (e.g. an
alkali for example sodium hydroxide); an adhesive entity such as,
for example, a muco- or bio-adhesive; excipients to maximize
solubility of active pharmaceutical compound(s); excipients to
maximize permeability of the active pharmaceutical compound(s) in
the GIT; an oil, for example a medium chain triglyceride
composition; a cationic lipid, for example a liposomal transfection
reagent; and/or a further surfactant.
[0221] The typical excipients for enhancing the permeability of the
epithelial barrier include but are not limited to sodium caprate,
sodium dodecanoate, sodium palmitate, SNAG, chitosan and
derivatives thereof, fatty acids, fatty acid esters, polyethers,
bile salts, phospholipids, alkyl polyglucosides, hydroxylase
inhibitors, antioxidants (e.g. ascorbic acid) and/or nitric oxide
donors, including nitric oxide or carbon dioxide donor groups
covalently attached to various active pharmaceutical ingredients.
The preceding list is of particular interest to enhance
permeability in the ileum.
[0222] To enhance permeability in the colon, typical excipients
include, but not limited to sodium caprate, sodium dodecanoate,
sodium palmitate, SNAG, chitosan and derivatives thereof, fatty
acids, fatty acid esters, polyethers, bile salts, phospholipids,
alkyl polyglucosides, hydroxylase inhibitors, antioxidants and/or
nitric oxide donors, including nitric oxide donor groups covalently
attached to various active pharmaceutical ingredients.
[0223] The composition may further comprise excipients to enhance
the therapeutic potential of active ingredients in the ileum and
colon including, but not limited to absorption limiters, essential
oils such as, for example, omega 3 oils, natural plant extracts
such as, for example, neem, ion-exchange resins, bacteria
degradable conjugation linkers such as, for example, azo bonds,
polysaccharides such as, for example, amylose, guar gum, pectin,
chitosan, inulin, cyclodextrins, chondroitin sulphate, dextrans,
guar gum and locust bean gum, nuclear factor kappa B inhibitors,
acids such as, for example, fumaric acid, citric acid and others,
as well as modifications thereof.
[0224] The composition may further comprise an oil, e.g. may
contain a single oil or a combination of oils, which may be any
pharmaceutically acceptable oil. Oils may include vegetable oils
(e.g. neem oil), petrochemical oils, and volatile essential oils.
The composition may comprise an oil selected from the group
consisting of: poly-unsaturated fatty acids such as, for example,
omega-3 oils; medium chain triglycerides; natural
triglyceride-based oils which include olive oil, sesame oil,
coconut oil, palm kernel oil, preferred include saturated coconut
and palm kernel oil-derived caprylic and capric fatty acids and
glycerine; other possible oils include linoleoyl macrogolglycerides
(polyoxylglycerides) such as, for example, Labrafil (e.g. product
number M2125CS by Gattefosse) and caprylocaproyl macrogolglycerides
such as, for example, Labrasol by Gattefosse.
[0225] As oils may be mentioned liquid lipids, for example selected
from medium chain triglyceride (MCT) compositions, the medium chain
triglyceride(s) being one or more triglycerides of at least one
fatty acid selected from C.sub.6-C.sub.12 fatty acids. It will be
understood that commercially available MCT compositions useful in
the invention are mixtures derived from natural products and
usually or always contain minor amounts of compounds which are not
MCTs; the term "medium chain triglyceride composition" is therefore
to be interpreted to include such compositions.
[0226] The composition of the invention may further comprise a
cationic lipid, for example a cationic liposomal transfection
reagent. Cationic lipids comprise a hydrophobic moiety, for example
an alkyl chain or alkenyl chain having one or more carbon-carbon
double bonds, and a cationic group. The hydrophobic group can be,
for example, a saturated alkyl chain or an unsaturated alkyl chain.
The cationic lipid may be selected from the group consisting of
DOTAP (N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane
methylsulfate), DOSPER (1,3-dioleoyloxy-2-(6-carboxyspermyl)-propyl
amide), DC-Cholesterol
HCl(3.beta.-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
hydrochloride), DODAP (1,2-dioleoyl-3-dimethylammonium-propane),
DDAB (Dimethyldioctadecylammonium), 12:0 EPC
(1,2-dilauroyl-sn-glycero-3-ethylphosphocholine), DOTMA
(1,2-di-O-octadecenyl-3-trimethylammonium propane), DOEPC
(1,2-dioleoyl-sn-glycero-3-ethylphosphocholine chloride salt), DOG
(1,2-dioleoyl-sn-glycerol), DODAP
(1,2-dioleoyl-3-dimethylammonium-propane), DOPE
(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC
(1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPG
(1,2-dioleoylsn-glycero-3-[phospho-rac-(1-glycerol)]sodium salt),
DOSPA
(2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N--N-dimethyl-1-propanam-
inium trifluoroacetate), DOTMA
(N-[1-(2,3-dioleyloxyl)propyl]-N,N,N-trimethylammonium chloride;),
DMRIE (1,2-dimyritsyloxypropyl-3-dimethylhydroxyethyl ammonium
bromide), DMKE (O,O'-dimyristyl-N-lysyl aspartate), DMKD
(O,O'-dimyristyl-N-lysyl glutamate), and DOPS
(1,2-dioleoyl-sn-glycero-3-[phospho-I-serine]sodium salt) and
combinations thereof.
[0227] The composition may further comprise a surfactant which is
envisaged to have a primary role other than micelle-formation and
selected from the group consisting of: cationic; amphoteric
(zwitterionic); anionic surfactants, including perfluoro-octanoate
(PFOA or PFO), perfluoro-octanesulfonate (PFOS), sodium dodecyl
sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfate
salts, sodium laureth sulfate, also known as sodium lauryl ether
sulfate (SLES) and alkyl benzene sulphonate; and non-ionic
surfactants including perfluorocarbons, polyoxyethyleneglycol
dodecyl ether (e.g. Brij such as, for example, Brij 35), Myrj (e.g.
Myrj 49, 52 or 59), Tween 20 or 80 (also known as Polysorbate)
(Brij, Myrj and Tween products are available commercially from
Croda), poloxamers which are nonionic triblock copolymers composed
of a central hydrophobic chain of polyoxypropylene (poly(propylene
oxide)) flanked by two hydrophilic chains of polyoxyethylene
(poly(ethylene oxide)). A preferred anionic surfactant in the
aqueous phase is SDS. Mixtures of further surfactants are also
contemplated, e.g. mixtures comprising perfluorocarbons.
[0228] In embodiments of the invention, the composition comprises a
hydrophilic surfactant which, without being bound by theory, is
believed at least partially to partition the aqueous phase (polymer
matrix).
[0229] Such surfactants intended for such inclusion in the aqueous
phase of the inventive composition are preferably readily diffusing
or diffusible surfactants to facilitate manufacturing and
processing of the composition of the invention. The surfactant may
have an HLB of at least 10 and optionally of at least 15, e.g. at
least 30 and optionally of 38-42, e.g. 40. Such surfactants can be
of any particular type (ionic, non-ionic, zwitterionic) and may
comprise as a proportion of dry weight of the composition from 0.1%
to 6%, e.g. 0.1% to 5%. 0.1% to 4% or 0.1% to 3%, more preferably
in a proportion of at least 1% and in particular between 1.0 and
4.5 or 5%, ideally within or just outside the 2-4% range, for
example from 2 to 3% or approximately 2% or approximately 4%.
[0230] Unless otherwise stated or required, all percentages and
ratios are by weight.
[0231] Particular anionic surfactants for inclusion in the aqueous
phase include perfluoro-octanoate (PFOA or PFO),
perfluoro-octanesulfonate (PFOS), sodium dodecyl sulfate (SDS),
ammonium lauryl sulfate, and other alkyl sulfate salts, sodium
laureth sulfate, also known as sodium lauryl ether sulfate (SLES)
and alkyl benzene sulphonate. A particular class of surfactant
comprises sulfate salts. A preferred anionic surfactant in the
aqueous phase is SDS. Mixtures of anionic surfactants are also
contemplated.
[0232] The physical form of the surfactant at the point of
introduction into the aqueous phase during preparation plays a role
in the ease of manufacture of the composition according to the
invention. As such, although liquid surfactants can be employed, it
is preferred to utilize a surfactant which is in solid form (e.g.
crystalline, granules or powder) at room temperature, particularly
when the aqueous phase comprises gelatin.
[0233] In general, mixtures of surfactants can be utilised e.g. to
achieve optimum long term stability of the composition of the
invention with shorter chain surfactants in general facilitating
shorter term stability (an aid to processing) and longer chain
surfactants facilitating longer term stability (an aid to shelf
life). In some embodiments, shorter chain surfactants have up to
C.sub.10 alkyl (e.g. C.sub.6-C.sub.10 alkyl) as the hydrophobic
portion of the surfactant whilst longer chain surfactants have
C.sub.10 or higher alkyl (e.g. C.sub.10-C.sub.22 alkyl) as the
hydrophobic portion of the surfactant. It is envisaged that
C.sub.10 alkyl surfactants may facilitate processing or facilitate
prolongation of shelf life, or both, depending on the identity of
the other excipients and of the active principle(s). Higher alkyl
may in particular implementations of the invention be
C.sub.11-C.sub.22 or C.sub.12-C.sub.22 alkyl, and in some
embodiments has a length of no greater than C.sub.18.
[0234] Instead of (or as complement to) the surfactant in the
aqueous phase, the invention also contemplates use of
surfactant-like emulsifiers (also known as crystallisation
inhibitors) such as, for example, HPMC (also known as hypromellose)
although their use is generally contemplated in relatively smaller
amounts to avoid high viscosity which may constrain processing
options.
[0235] The composition may further comprise excipients or other
active pharmaceutical or other ingredients to enhance systemic
bioavailability following absorption in the GIT, such as the small
intestine, including efflux pump inhibitors, including, but not
limited to PgP pump inhibitors, and metabolism inhibitors,
including, but not limited to, cytochrome P450 3A inhibitors.
[0236] The composition may further comprise excipients to reduce
systemic side effects associated with absorption in the GIT, such
as the small intestine, including, but not limited to,
antioxidants, such as, for example, curcuminoids, flavanoids or
more specifically including curcumin, beta-carotene,
.alpha.-tocopherol, ascorbate or lazaroid.
[0237] The composition may further or separately comprise
antioxidants (such as, for example, ascorbic acid or BHT--butyl
hydroxy toluene) taste-masking or photosensitive components or
photoprotective components. Antioxidants may be incorporated in the
aqueous phase (e.g. hydrophilic antioxidants) or in the surfactant
phase (e.g. hydrophobic antioxidants such as, for example, vitamin
E) for example up to 1% by weight, preferably between 0.01 and
0.50% by weight, more preferably between 0.10 to 0.20% by
weight.
[0238] The composition may further comprise immune-enhancing
nutrients such as Vitamins A/B/C/E; Carotenoids/beta-carotene and
Iron, Manganese, Selenium, Zinc. Such nutrients may be present in
composition, or if the composition has a coating, for example if it
is the form of a bead, the nutrients may be included in the
coating.
[0239] The composition may further or separately include an
adhesive to ensure that if desired the bead of the dosage form
remain, or remain for longer, in the gastric environment. Beads
according to the invention may also comprise materials facilitating
or enabling floating or density reduction e.g. as a means of
localising beads in desired GI sites. The bead of the dosage form
may also have the means to swell and/or aggregate in the stomach or
other GI site.
[0240] The dosage form of the invention may comprise the excipients
disclosed above. In certain embodiments any excipients present in
the dosage form may not be contained within the population of the
composition of the dosage form.
Shape, Size and Geometry
[0241] The composition of the invention can be formed into a
limitless number of shapes and sizes. In the section below
describing the process for making the composition, various methods
are given including pouring or introducing a fluid micelle
dispersion into a mould where it hardens or can be caused to
harden. Thus the composition can be created in whichever form is
desired by creating an appropriate mould (e.g. in the shape of a
disc, pill or tablet). However, it is not essential to use a mould.
For example, the composition may be formed into a sheet e.g.
resulting from pouring a fluid micelle dispersion onto a flat
surface where it hardens or can be caused to harden.
[0242] Preferably, the composition may be in the form of spheres or
spherical-like shapes made as described below. Preferably, the
composition of the invention is in the form of substantially
spherical, seamless beads. The absence of seams on the bead surface
is an advantage e.g. in further processing, for example coating,
since it allows more consistent coating, flowability etc. The
absence of seams on the beads also enhances consistency of
dissolution of the beads.
[0243] The preferred size or diameter range of beads according to
the invention can be chosen to avoid retention in the stomach upon
oral administration of the beads. Larger dosage forms are retained
for variable periods in the stomach and pass the pyloric sphincter
only with food whereas smaller particles pass the pylorus
independently of food. Selection of the appropriate size range (see
below) thus makes the prediction of therapeutic effect post-dosing
more accurate. Compared to a single large monolithic oral format
such as, for example, a traditional compressed pill, a population
of beads released into the GI tract (as foreseen by the dosage form
of the present invention) permits greater intestinal lumen
dispersion so enhancing absorption via exposure to greater
epithelial area, prevents irritation (e.g. as otherwise seen with
NSAIDs) and achieves greater topical coating (e.g. as may be
desired for local drug effect in certain parts of the GI tract for
example the colon). Reduction of residence time in the ileo-caecal
junction is another potential advantage.
[0244] The composition of the invention is preferably monolithic
meaning internally (i.e. cross-sectionally) homogeneous, excluding
a possible thin skin of matrix material and excluding any coating
layers.
[0245] The beads provided for by the composition of the present
invention generally range in diameter from 0.5 mm to 10 mm with the
upper limit preferably 5 mm, e.g. 2.5 mm A particularly convenient
upper limit is 2 mm or 1.7 mm. The lower limit can preferably be 1
mm, e.g. 1.2 mm, more preferably from 1.3 mm, most preferably from
1.4 mm. In one embodiment the diameter is from 0.5 to 2.5 mm, for
example from 1 mm to 3 mm, 1 mm to 2 mm, 1.2 mm to 3 mm or 1.2 mm
to 2 mm. The beads may have a diameter of no more than 2.5 mm,
irrespective of their minimum size. The beads may have a diameter
of no more than 2 mm, irrespective of their minimum size.
[0246] A bead as described herein may have an aspect ratio of no
more than 1.5, e.g. of no more than 1.3, for example of no more
than 1.2 and, in particular, of from 1.1 to 1.5, 1.1 to 1.3 or, 1.1
to 1.2. A population of beads as described herein, e.g. at least 10
beads, may have an average aspect ratio of no more than 1.5, e.g.
of no more than 1.3, for example of no more than 1.2 and, in
particular, of from 1 to 1.5, 1 to 1.3 or 1 to 1.2. The aspect
ratios mentioned in this paragraph optionally apply to coated beads
and optionally apply to uncoated beads. Average aspect ratio is
suitably determined for a population of beads, e.g. at least 10
beads, using a particle size analyser, for example an Eyecon.TM.
particle characteriser of Innopharma Labs, Dublin 18, Ireland.
[0247] The beads of the disclosure may, therefore, have a size as
disclosed in paragraph [00194] above and an aspect ratio of from 1
to 1.5. The beads of the disclosure may have a size as disclosed in
paragraph [00194] above and an aspect ratio of no more than 1.3,
for example of no more than 1.2 and, in particular, of from 1.1 to
1.5, 1.1 to 1.3 or, 1.1 to 1.2.
[0248] Bead size (diameter) may be measured by any suitable
technique, for example microscopy, sieving, sedimentation, optical
sensing zone method, electrical sensing zone method or laser light
scattering. For the purposes of this specification, bead size is
measured by analytical sieving in accordance with USP General Test
<786> Method I (USP 24-NF 18, (U.S. Pharmacopeial Convention,
Rockville, Md., 2000), pp. 1965-1967).
[0249] In embodiments, beads of the invention are monodisperse. In
other embodiments, beads of the invention are not monodisperse. By
"monodisperse" is meant that for a population of beads (e.g. at
least 100, more preferably at least 1000) the beads have a
coefficient of variation (CV) of their diameters of 35% or less,
optionally 25% or less, for example 15% or less, such as e.g. of
10% or less and optionally of 8% or less, e.g. 5% or less. A
particular class of polymer beads has a CV of 25% or less. CV when
referred to in this specification is defined as 100 times (standard
deviation) divided by average where "average" is mean particle
diameter and standard deviation is standard deviation in particle
size. Such a determination of CV is performable using a sieve.
[0250] The invention includes beads having a CV of 35% and a mean
diameter of 1 mm to 2 mm, e.g. 1.5 mm. The invention also includes
beads having a CV of 20% and a mean diameter of 1 mm to 2 mm, e.g.
1.5 mm, as well as beads having a CV of 10% and a mean diameter of
1 mm to 2 mm, e.g. 1.5 mm. In one class of embodiments, 90% of
beads have a diameter of from 0.5 mm to 2.5 mm, e.g. of from 1 mm
to 2 mm.
[0251] Another possible form of the composition of the invention is
as hemispherical beads two of which may optionally be joined at the
flat face to create a single bead with two distinct halves, each
having a distinct composition, if that is desired, e.g. each
containing different active principles or the same active
principles but different excipients e.g. to achieve differing
permeability, solubilisation or release profiles as between the two
hemispheres.
[0252] The bead provided for by the composition of the invention,
may also be used as a starting point for creation of further e.g.
pharmaceutical or nutraceutical forms for example by using the bead
as a nonpareil seed on which additional layers of material can be
applied as is well known to a person skilled in the art e.g. of
pharmaceutical science. The material of the additional layers may
comprise the same or different active principle and/or the same or
different excipients as are described in this document. Such
variants allow differential release of the same or different active
principles and facilitate inclusion of multiple fixed-dose
combination products as for example discussed in connection with
the popularly termed "polypill" which denotes a single pill
comprising more than one active principle in a fixed dose
combination, an idea of particular relevance to cardiovascular
medicine.
[0253] The composition of the invention may have a coat of
additional material on its outer surface. This coat may be applied
in a number of ways, including drug layering, as described more
particularly in the section below entitled "coating". In one such
embodiment, the composition of the invention comprises an acid e.g.
included within the hydrogel-forming polymer matrix or as a liquid
core in mini-capsular format and bicarbonate applied as a coat e.g.
by drug layering. If the composition has a polymeric coat, e.g. to
control release into the colon, the bicarbonate may optionally or
additionally be included in or be absent from the coating polymer.
This composition is intended to release carbon dioxide in the GI
tract e.g. to reduce pain or to reduce inflammation. In a related
embodiment, the core or the composition comprises an acid to
enhance the solubility of active principles of various pKa (acid
dissociation constant) in the small intestine or colon.
Alternatively, the core or the composition comprises a base to
enhance the solubility of active principles of various pKa in the
stomach.
Other Characteristics
[0254] The composition of the invention, in certain embodiments,
comprises one or more elements, components, excipients, structural
features, functional features or other aspects of the prior art
described above.
[0255] To summarise a limited number of embodiments of the
invention, the composition as described above and elsewhere herein
may additionally be one or more of the following: substantially
water-free, in a gel state, in a solid state, undissolved,
non-powdered, formed, shaped, and not in solution.
[0256] It is preferable that the composition of the invention is
essentially or substantially dry, e.g. contains less than 5%,
preferably less than 1% of free water by weight. The beads of the
composition are preferably homogeneous although processing
conditions may be varied (see below) to achieve for example
heterogeneity such as, for example, a harder skin and softer core
with less than complete immobilization of the micelles towards the
core as opposed to the surface of the bead. Larger forms or shapes
of the bead according to the invention may particularly be
engineered to embody such heterogeneity.
[0257] The low free-water content is a distinguishing feature of
certain embodiments of the compositions of the present invention.
The free-water content can be measured using thermogravimetic
analysis (TGA), for example with commercially available
instrumentation, e.g. using a TGA Q 500 of TA Q series instrument.
TGA measures changes in weight in relation to a change in
temperature. For example, a TGA method can comprise a temperature
scan, e.g. from 20 to 400.degree. C. at 20.degree. C. per minute,
where the moisture content is obtained from the sample weight loss
at about 100 degrees Celsius.
[0258] In one embodiment, the micelle dispersion is homogeneously
dispersed in the solidified hydrogel-forming polymer with
substantial absence of coalescence between adjacent micelles. Thus
the micelle dispersion is preferably maintained during
solidification.
[0259] The composition of the invention generally comprises
multiple micelles within a moulded or shaped form which might
typically contain many hundreds or thousands of micelles as
distinct from a powder which generally derives from micron-sized
particles incorporating a single or a small number of micelles
often following agglomeration of the micelles during spray-drying.
While powder embodiments are not excluded, the composition of the
invention, if particulate, preferably comprises particles larger
than powder particles such that the composition is in a
non-powdered form.
[0260] The "solid" composition of the invention (i.e. after
solidification and drying of the hydrogel in the processes
described below) is suitably such that the constituents readily
form micelles in at least one of, e.g. in both of, the
surfactant-containing small intestine and the surfactant-limited
colon.
[0261] In certain embodiments the composition of the invention
comprises: [0262] a) 25 to 70% (suitably 40 to 65% and particularly
45 to 60%) hydrogel-forming polymer (for example gelatin); [0263]
b) 20 to 50% (suitably 30 to 40%) surfactant (for example Kolliphor
HS 15); [0264] c) 1 to 10% (suitably 3 to 8%) plasticiser (for
example sorbitol); and [0265] d) 0.01 to 2% (suitably 0.1 to 1% or
0.1 to 0.3%) adjuvant (for example .alpha.-GalCer); wherein the %
are by weight based on the dry weight of the composition in the
absence of any coating on the composition. The composition in these
embodiments further comprises a microorganism selected from live,
killed, attenuated and inactivated microorganism. At a
concentration to give the desired dosage in the final dosage form,
for example sufficient to provide 10.sup.8 to 10.sup.14 cells per
dose.
[0266] In one embodiment, the invention allows for beads or other
shaped units having immediate release (IR) characteristics e.g.
bearing no coat, enteric-only coat or coat designed to prevent
release and/or dissolution of the bead only for a limited time or
lacking a retardant in the aqueous phase. In another embodiment,
the invention allows for beads having delayed or sustained release
(SR) characteristics e.g. bearing a coat (or more than one coat) as
described in more detail below, particularly in the section
entitled "coating". The invention also provides for an embodiment
in which immediate release beads are produced in combination with a
Sustained Release or Controlled Release (CR) beads in varying
ratios of IR:SR/CR. The immediate release beads can be combined
with a Sustained or Controlled release bead component in the
following ratios (w/w by potency) e.g. 10% Immediate Release
(IR)+90% Sustained (SR)/Controlled Release (CR) minicapsules; 20%
IR+80% SR/CR; 30% IR+70% SR/CR; 40% IR+60% SR/CR and 50% IR+50%
SR/CR.
[0267] In embodiments, the beads or shaped units have an immediate
release coat and, between the core made of the micelle-containing
composition and the IR coat, a sub-coat to do at least one of the
following, amongst others: provide mechanical strength; prevent
moisture absorption; modulate release of active agent from the
core; stabilise release of active agent from the core (e.g.
modulate and stabilise release of active agent from the core).
[0268] In embodiments the self-assembly structures for example
micelles, are characterised by the size of the self-assembly
structure. A convenient method to determine micelle size is dynamic
light scattering as hereinbefore described, wherein the micelle
size is determined as a hydrodynamic diameter. Light scattering is
a technique which can be used to determine the size distribution
profile of particles in solution. When light hits particles the
light scatters in all directions (Rayleigh scattering). If the
light source is a laser, and thus is monochromatic and coherent,
then a time-dependent fluctuation in the scattering intensity is
observed. These fluctuations are due to the fact that the particles
in solutions are undergoing Brownian motion and so the distance
between the scattering particles in solution is constantly changing
with time. The dynamic information of the particles is derived from
an autocorrelation of the intensity trace recorded during the
experiment which is dependent on measured time delays. At short
time delays, the correlation is high because the particles do not
have a chance to move to a great extent from the initial state that
they were in. As the time delays become longer, the correlation
starts to exponentially decay to zero, meaning that after a long
time period has elapsed, there is no correlation between the
scattered intensity of the initial and final states. This
exponential decay is related to the motion of the particles,
specifically to the diffusion coefficient. To fit the decay,
numerical methods are used, based on calculations of assumed
distributions. If the sample is monodisperse then the decay is
simply a single exponential and the polydispersity index (PdI)
would be zero or close to it; however, if the sample is
polydisperse the decay can be bi-exponential (when there are two
populations), or have an even more complex decay. The
polydispersity index is obtained from the fitting of the decay and
will reach its maximum at PdI equal one.
[0269] Other useful characterising methods for measuring the size
and formation of self-assembly structures such as micelles include
Small angle X-ray scattering and Diffusion Nuclear Magnetic
Resonance. Such techniques are well known, see for example Oliver
et al PLOS one, May 2013, Vol 8 (5), e62488 and Colafemmina et al,
J. Phys Chem B 2007, 111, 7184-7193.
Manufacturing Processes
[0270] The manufacturing processes described herein comprise mixing
of liquids. Such mixing processes must be performed at temperatures
at which the substances to be mixed in the liquid state are in
liquid form. For example, thermoreversible gelling agents must be
mixed at a temperature where they are in the liquid state, for
example at a temperature of 50 to 75.degree. C., for example 50 to
70.degree. C., or 55-75.degree. C., e.g. 60-70.degree. C. and in
particular embodiments about 55.degree. C. or 65.degree. C. in the
case of mixing compositions comprising aqueous gelatin. Kolliphor
HS 15 is also to be mixed in the liquid state and is suitably
maintained at room temperature or slightly higher, for example
maintained at a temperature of at least 30.degree. C. for that
purpose, e.g. of 35-50.degree. C. and in particular 40.degree. C.;
where both Kolliphor HS 15 and aqueous gelatin are to be mixed,
then a higher temperature, e.g. of 50-75.degree. C., for example
55-75.degree. C., is used at which Kolliphor HS 15 is liquid as
well as aqueous gelatin.
[0271] Compositions as disclosed herein may be made by mixing
materials comprising water, a hydrogel-forming polymer, a
surfactant, and an active ingredient(s) to form a self-assembly
structure dispersion within an aqueous phase comprising the
hydrogel-forming polymer. The hydrogel-forming polymer is then
caused or allowed to gel. Suitably, the process includes
formulating or processing the aqueous self-assembly structure
dispersion into a desired form, e.g. a bead, which forming process
may comprise moulding but preferably comprises ejecting the aqueous
micelle dispersion through a single orifice nozzle to form droplets
which are caused or allowed to pass into a cooling medium, e.g. a
water-immiscible cooling liquid, in which the droplets cool to form
for e.g. beads.
[0272] The mixing of the materials may comprise mixing an aqueous
premix (or aqueous phase) and a surfactant premix (or surfactant
phase), wherein the aqueous premix comprises water and
water-soluble substances whilst the surfactant premix comprises
surfactant and surfactant-soluble substances. In some embodiments
the aqueous premix comprises at least one water-dispersible
substance. In some embodiments the surfactant premix comprises at
least one surfactant-dispersible substance.
[0273] The aqueous premix comprises, or usually consists of, a
solution in water of water-soluble constituents, namely the
hydrogel-forming polymer, any water-soluble excipient(s), any
hydrophilic active(s). The aqueous premix may include at least a
portion of, e.g. all of, the microorganism content of the final
composition. The aqueous premix may include at least a portion of,
e.g. all of, the adjuvant content of the final composition. The
aqueous premix may include a plasticiser, i.e. a water-soluble
excipient, as described elsewhere in this specification. The
aqueous premix may include a surfactant, e.g. to increase polymer
viscosity and improve emulsification and thereby help prevent
precipitation of active agent during processing. SDS is an example
of such a surfactant. The aqueous phase may include one or more
controlled release polymers. In any event, the constituents of the
aqueous premix may be agitated for a period of, for example, from 1
hour to 12 hours to form the completed aqueous premix.
[0274] The surfactant phase premix comprises a solution in a
described surfactant of hydrophobic and amphiphilic constituents.
The specification hereby discloses surfactant phase premixes which
include at least a portion of the microorganism content and at
least a portion of the adjuvant content of the final composition.
The surfactant phase premix may include all of the microorganism
content and at least a portion of, e.g. all of, the adjuvant
content of the final composition. The surfactant phase premix may
include a portion of the microorganism content of the final
composition, a portion also being included in the aqueous phase
premix, and at least a portion of, e.g. all of, the adjuvant
content of the final composition. Where the surfactant phase
includes a portion of the microorganism content, the portion may be
at least 50 wt %, e.g. at least 75 wt %. Where the surfactant phase
includes a portion of the adjuvant content, the portion may be at
least 50 wt %, e.g. at least 75 wt %. It will be recalled that the
surfactant may comprise a hydrophobic chain and a hydrophilic
chain. The hydrophobic and amphiphilic constituents, if any, may
comprise one or more active ingredients selected from hydrophobic
and amphiphilic active ingredients.
[0275] The surfactant premix, therefore, will in many cases include
a microorganism. Typically, the microorganism will be included
directly into the surfactant, for example as a lyophilisate or
other dry powder, or the microorganism may be included in the
surfactant as an aqueous suspension. The microorganism may be
included in the surfactant both as a lyophilisate or other dry
powder and as an aqueous suspension. The invention therefore
provides a process for manufacturing a surfactant/active premix. A
process of the invention comprises mixing (i) a surfactant, and
(ii) a microorganism, and optionally (iii) an adjuvant. The
surfactant premix may comprise additional substances to the
surfactant and any actives. For example it may comprise additional
excipients. Such additional excipients may be hydrophobic or
amphiphilic, for example they may comprise a water-immiscible
material, e.g. an oil. An additional excipient may therefore be a
liquid lipid, for example a medium chain triglyceride (MCT)
composition, the medium chain triglyceride(s) being one or more
triglycerides of at least one fatty acid selected from
C.sub.6-C.sub.12 fatty acids. Any one or more active agents may be
pre-dissolved in a solvent, e.g. ethanol or an MCT composition,
before being combined into the surfactant premix. In some
embodiments, the components of the surfactant premix are mixed (or
otherwise agitated) for a period of, for example, 10 minutes to 3
hours to form the premix.
[0276] The two premixes may be combined and agitated, for example
for a period of a few seconds to an hour, for example from 30
seconds to 1 hour, suitably 5 mins to an hour, to form a dispersion
of self-assembly structures (e.g. micelles) in an aqueous
hydrogel-forming polymer, which dispersion may then be further
processed to form the final formulation. The two premixes may be
combined into the dispersion by agitation in a mixing vessel; they
may additionally or alternatively be combined in a continuous flow
mixer.
[0277] The basic method for making the composition of the
invention, therefore, is to mix a fluid form (preferably a
solution) of the hydrogel-forming polymer (or mixture of polymers)
with the active ingredient(s) and with the surfactant to form a
dispersion in the hydrogel formed by the polymer. Taking account of
the final composition required (as described elsewhere herein), the
surfactant and the fluidic hydrogel-forming polymer (i.e. the
solution or suspension of hydrogel-forming polymer) may be mixed in
a proportion in the range 1:2-5, preferably approximately 1:3 or
1:4. In general, only gentle stirring of the components is required
using a magnetic or mechanical system e.g. overhead stirrer as
would be familiar to a person skilled in the art to achieve a
dispersion of self-assembly structures, e.g. micelles. Continuous
stirring is preferred. Mixing may also be achieved using an in-line
mixing system. Any appropriate laboratory stirring apparatus or
industrial scale mixer may be utilized for this purpose for example
the Magnetic Stirrer (manufactured by Stuart) or Overhead Stirrer
(by KNF or Fisher). It is preferred to set up the equipment in such
a way as to minimise evaporation of contents such as, for example,
water. In one embodiment of the process of the invention, it is
preferred to utilise a closed system for stirring in order to
achieve this aim. In-line mixing may be particularly suitable for
closed system processing.
[0278] The invention includes embodiments in which the surfactant
phase liquid and water-insoluble constituents are formed into a
clear solution which is mixed with the aqueous phase. Both phases
may be clear solutions prior to mixing of them to form an
emulsion.
[0279] In embodiments the concentration of the surfactant in the
surfactant pre-mix is selected such that upon combination with the
aqueous pre-mix the surfactant concentration in the combined
mixture exceeds the CMC for the surfactant used such that micelles
are formed in the aqueous phase comprising the hydrogel-forming
polymer. Depending on the concentration of surfactant used
self-assembly structures other than micelles may also form. The CMC
for a particular surfactant may be determined using well known
methods, for example as described in Surfactants and Polymers in
Aqueous Solutions Second Edition, Chapter 2, Holmberg et al.
[0280] In embodiments mixing of the aqueous and surfactant
pre-mixes results in the formation of a clear solution, for example
a microemulsion, in which the aqueous phase comprising the
hydrogel-forming polymer is the continuous phase. The formation of
a clear solution upon mixing of the surfactant phase and aqueous
phase is generally indicative that very small self-assembly
structures have formed, for example as a microemulsion.
Microemulsions are a thermodynamically stable dispersion of the
self-assembly structures in the aqueous phase, the size of the
self-assembly structures being sufficiently small to give a
transparent appearance. As hereinbefore mentioned, in embodiments
the size of the self-assembly structures present as the disperse
phase resulting from the mixing of the aqueous and surfactant
phases is about 0.5 nm to 200 nm, for example about 1 nm to 50 nm,
or about 5 nm to 25 nm. The size of the self-assembly structures
formed and other characteristics such as the optical isotropicity
of the composition (for example a microemulsion) may be determined
using well known techniques as hereinbefore described.
[0281] In the embodiment where the polymer matrix substantially
consists of gelatin with the addition of sorbitol, the aqueous
phase of polymer matrix is prepared by adding the appropriate
quantities of sorbitol (and surfactant if desired) to water,
heating to approximately 50 to 75.degree. C., for example
60-75.degree. C. until in solution and then adding gelatin although
the precise order and timing of addition is not critical. A typical
"gelatin solution" comprises 8 to 25%, (for example 15-25%,
preferably 17-18%) gelatin; 75%-85% (preferably 77-82%) of water
plus from 1-5% (preferably 1.5 to 3%) sorbitol.
[0282] The choice of temperature at which the dispersion is formed
depends however on various factors including the temperature
lability of the active ingredient and the amount of plasticiser
included in the gelatin, the type of gelatin, as well as other
factors. Generally however, the gelatin solution (especially in the
case of standard or normal gelatin) is maintained at 50 to
70.degree. C., suitably 60.degree. C.-70.degree. C. to maintain it
in a fluid state.
[0283] The processing temperature can however be reduced to a
desirable target temperature e.g. 37.degree. C. by use of lower
melting-point gelatin (or gelatin derivatives or mixtures of
gelatins with melting point reducers) or other polymer matrix
material such as, for example, sodium alginate. Alternatively,
temperature-labile active principles may be processed at higher
temperatures by using appropriate apparatus or machinery which
limits the time during which the temperature-labile active
principle is in contact with the higher temperature medium. For
example, if gelatin droplets are being formed by machine extrusion
and immediately cooled e.g. in a cooling bath, additional
appropriate inlet tubing can be used to introduce
temperature-sensitive active principle into the fluid gelatin
solution (and the mixture can be immediately homogenized) very
shortly before ejection from a beading nozzle or other dropletting
process such that the duration of exposure of the active principle
to the higher temperature gelatin is limited so reducing the degree
of any heat-dependent degradation of the active ingredient. This
process may use any appropriate device such as, for example, a
homogenizer, e.g. a screw homogenizer, in conjunction with an
extrusion-type apparatus as described for example in WO 2008/132707
(Sigmoid Pharma) the entirety of which is incorporated herein by
reference.
[0284] The invention therefore includes processes in which the
mixture of the aqueous and surfactant phases is ejected through a
single orifice nozzle to form droplets, the hydrogel-forming
polymer then being caused or allowed to solidify whereby the
droplets form beads, and wherein the hydrogel-forming polymer is a
thermotropic polymer or a mixture of thermotropic polymers, the
aqueous phase (also called aqueous premix) being at an elevated
temperature and the surfactant phase (also called surfactant
premix) being at a lower temperature suitably not exceeding ambient
temperature, the two premixes flowing through respective feed lines
to a mixing apparatus where the two premixes are mixed, and wherein
at least one of the two premixes travels a greater distance through
its feedline than the mixture does in travelling from the mixing
apparatus to the nozzle. The two phases may be mixed at a position
juxtaposed to the nozzle, e.g. by in-line mixing apparatus
juxtaposed to the nozzle.
[0285] The invention includes processes in which the surfactant
premix is at a lower temperature than the aqueous premix prior to
mixing the two premixes. Maintaining the surfactant premix at a
lower temperature than the aqueous premix can be utilised to reduce
damage to heat labile components of the composition (for example
the microorganism and/or adjuvant) during the process. In
embodiments it may be advantageous to form the mixture of the
surfactant and aqueous premixes adjacent to the nozzle. Such an
arrangement enables the mixture to be immediately ejected from the
nozzle (generally a single nozzle) as droplets into a cooling
medium, thereby minimising the time that the components of the
surfactant premix (which may contain heat labile substances such as
an antigen or adjuvant) are exposed to elevated temperature. The
time that the components of the surfactant premix are exposed to
elevated temperature during the process can be further minimised by
locating the exit of the mixing apparatus (eg in-line mixer) in
close proximity to the nozzle and/or using a high mixture flow rate
from the mixing apparatus to the nozzle. The cooling medium may be
as herein described, for example a low temperature gas or suitable
liquid medium such as an oil bath.
[0286] Generally, where the self-assembly structure-forming
surfactant is a liquid there is no need to heat it and the active
ingredient is added at room temperature with stirring until clear.
It is possible that the surfactant phase may comprise additional
components. These other components may include a volatile (or
non-volatile) solvent in addition to the surfactant. The surfactant
phase may also contain the appropriate amount of active ingredient
(if any is added to the surfactant prior to mixing the surfactant
with the aqueous phase) to achieve the target proportion of active
ingredient as described elsewhere herein and in the examples. In
the embodiments where the surfactant is a waxy solid such as, for
example, Kolliphor HS 15 it is appropriate to heat the waxy solid,
e.g. to above 30.degree. C., to provide a liquid.
[0287] The dispersion is formed by addition of the surfactant to
the liquid aqueous phase with stirring as described above. The
resultant dispersion then has the composition of the solidified
beads described above but with liquid water still present.
[0288] The active ingredient(s) may optionally be added after
mixing the aqueous phase and surfactant.
[0289] The self-assembly structure dispersion is then poured or
introduced into a mould or other vessel or poured onto sheets or
between sheets or delivered dropwise (or extruded) into another
fluid such that the polymer matrix-containing aqueous phase, on
solidification, takes the form of the mould, vessel, sheet or
droplet/bead intended. It is preferred to progress to mould-forming
e.g. beading without delay.
[0290] Alternatively to moulding, specialised or customised
machinery can be employed for example to create the hemispherical
beads described above (see section above entitled "Shape, Size and
Geometry") in which the invention takes the form of hemispherical
beads. It is possible to manufacture a single bead made from
joining two such hemispheres (i.e. a single bead having two
distinct halves) by using specialist apparatus in which two tubes
through which two different emulsions are flowing, normally of
circular cross section, are joined shortly before an extrusion
point or nozzle (which may be vibrating) into a single dual lumen
tube with a flat wall separating the two emulsion flows and which
prevents the two emulsions from coming into contact until the point
of extrusion. The cross-section of the joined dual-lumen tube up to
the point of extrusion therefore appears as two semicircles. In
operation, the two hemispherical emulsion flows combine to form a
single, substantially spherical, bead on extrusion such that normal
droplets are ejected/extruded for solidification.
[0291] Solidification can occur in a variety of ways depending on
the polymer of the matrix, for example by changing the temperature
around the mould, vessel, sheet, droplet/bead etc or by applying a
solidification fluid or hardening solution so that the moulded
shape is gelled or solidified. In certain embodiments both
temperature change and application of a solidifying fluid or
hardening solution are employed together or simultaneously.
[0292] In the preferred embodiment in which the composition of the
invention takes the form of beads, the beads may be formed for
example by dropping the self-assembly structure dispersion dropwise
into a fluid which effects solidification. Where the viscosity of
the emulsion to be beaded reaches a certain point, drop formation
becomes more difficult and specialised apparatus is then
preferred.
[0293] By use of the term "dry", it is not sought to imply that a
drying step is necessary to produce the dry micelle dispersion
(although this is not excluded) rather that the solid or solidified
aqueous external phase is substantially free of water or free of
available water. Solidification of the aqueous phase (external
phase) may have arisen through various means including chemically
(e.g. by cross-linking) or physically (e.g. by cooling or heating).
In this respect, the term "aqueous phase" is nevertheless employed
in this document to denote the external (continuous) phase of the
bead of the invention even though water, in certain embodiments, is
largely absent from (or trapped within the cross-linked matrix of)
the bead of the invention. The external phase of the composition of
the invention is however water-soluble and dissolves in aqueous
media. In one embodiment, self-assembly structures are released
when the aqueous phase dissolves or is exposed to aqueous media,
irrespective of the form adopted by the micelle-forming surfactant
in the solid composition.
[0294] Accordingly, in some embodiments of the invention the
composition according to the invention releases self-assembly
structures upon dissolution or exposure to an aqueous medium, for
example a dissolution medium such as gastro-intestinal fluid
following oral administration of the composition. In embodiments
the self-assembly structures released from the composition (for
example micelles) are about 0.5 nm to 200 nm for example from about
1 nm to 50 nm, or about 5 nm and 25 nm. The size of the
self-assembly structure (eg micelle) may be determined by for
example dynamic light scattering as hereinbefore defined. Suitably,
the release of such self-assembly structures (eg micelles) from a
composition according to the invention may be determined by placing
the composition in an aqueous dissolution medium and measuring the
size of the self-assembly structures released into the dissolution
medium using dynamic light scattering. The dissolution medium
should be such that the matrix comprising the hydrogel forming
polymer is exposed to the aqueous medium to allow the formation
and/or release of self-assembly structures (for example micelles)
into the dissolution medium. Accordingly, when the composition is
coated to delay or control the release it may be necessary to
adjust for example the pH of the medium or the residence time in
the dissolution medium to enable the dissolution medium to
penetrate into the core comprising the hydrogel matrix and allow
the formation and/or release of the self-assembly structures into
the dissolution medium. For example, when a composition is coated
with an enteric coating the pH of the dissolution medium may need
to be increased to pH>5.5 or >6.5 to allow dissolution of the
enteric coating and exposure of the core to the aqueous dissolution
medium. The dissolution medium used to measure the self-assembly
structures may simply be water, for example water at pH 7.2-7.3 at
37.degree. C. After placing the composition into the dissolution
medium the dissolution medium may be regularly sampled and analysed
for the presence/release of self-assembly structures from the
composition using for example the dynamic light scattering methods
described herein.
[0295] In the case where solidification can be achieved by raising
or reducing temperature, the temperature of the solidification
fluid can be adapted to achieve solidification at a desired rate.
For example, when gelatin is used as the hydrogel-forming polymer,
the solidification fluid is at a lower temperature than the
temperature of the emulsion thus causing solidification of the
polymer matrix. In this case, the solidification fluid is termed a
cooling fluid.
[0296] In the case where solidification can be achieved chemically,
e.g. by induction of cross-linking on exposure to a component of
the solidification fluid, the concentration of such component in
the solidification fluid and/or its temperature (or other
characteristic or content) can be adjusted to achieve the desired
rate and degree of solidification. For example, if alginate is
chosen as the polymer matrix, one component of the solidification
fluid may be a calcium-containing entity (such as, for example,
calcium chloride) able to induce cross-linking of the alginate and
consequent solidification. Alternatively, the same or similar
calcium-containing entity may be included (e.g. dispersed) in the
aqueous phase of the fluid emulsion prior to beading and triggered
to induce cross-linking e.g. by applying a higher or lower pH to a
solidification fluid into which droplets of emulsion fall dropwise
or are introduced. Such electrostatic cross-linking can be varied
as to the resulting characteristics of the bead by control of
calcium ion availability (concentration) and other physical
conditions (notably temperature). The solidification fluid may be a
gas (for example air) or a liquid or both. For example, when
gelatin is used as the hydrogel-forming polymer matrix, the
solidification fluid can be initially gaseous (e.g. droplets
passing through cooling air) and then subsequently liquid (e.g.
droplets passing into a cooling liquid). The reverse sequence may
also be applied while gaseous or liquid cooling fluids alone may
also be used. Alternatively, the fluid may be spray-cooled in which
the emulsion is sprayed into a cooling gas to effect
solidification.
[0297] In the case of gelatin or other water-soluble polymer (or
polymer mixture) destined to form the immobilization matrix, it is
preferred that the solidification fluid be a non-aqueous liquid
(such as, for example, medium chain triglycerides, mineral oil or
similar preferably with low HLB to ensure minimal wetting) which
can conveniently be placed in a bath (cooling bath) to receive the
droplets of micelle dispersion as they solidify to form beads. Use
of a non-aqueous liquid allows greater flexibility in choice of the
temperature at which cooling is conducted.
[0298] Where a liquid cooling bath is employed, it is generally
maintained at less than 20.degree. C., preferably maintained in the
range 5-15.degree. C., more preferably 8-12.degree. C. when
standard gelatin is used as the hydrogel-forming polymer. If a
triglyceride is chosen as the cooling fluid in the cooling bath, a
preferred example is Miglyol 810 from Sasol.
[0299] If gelatin or another thermotropic polymer or polymer
mixture is selected as the hydrogel-forming polymer matrix, respect
for appropriate temperature ranges ensures solidification of the
polymer at an appropriate rate to avoid destruction e.g. of
tertiary protein structure in the case where the active principle
is a protein.
[0300] If alginate is selected as the polymer matrix, a typical
method of making beads involves dropwise addition of a 3% sodium
alginate solution in which oil droplets are dispersed as described
above into a 4.degree. C. crosslinking bath containing 0.1 M
calcium chloride to produce calcium alginate (this method can be
referred to as "diffusion setting" because the calcium is believed
to diffuse into the beads to effect cross-linking or setting).
Using a syringe pump, or Inotech machine, droplets can be generated
or extruded (egg at 5 mL/h if a pump is used) through a sterile
needle or other nozzle (described elsewhere herein) which can be
vibrating as discussed elsewhere herein. Airflow of between 15 and
20 L/min through 4.5 mm tubing can be applied downwards over the
needle to reduce droplet size if desired. Newly formed beads can
then be stirred in the calcium chloride bath for up to an hour. If
carrageenan is used as the polymer matrix both salt and reduction
in temperature e.g. by dropping into cooling oil may be used to
obtain solidification.
[0301] An alternative approach when using alginate is internal
gelation in which the calcium ions are dispersed in the aqueous
phase prior to their activation in order to cause gelation of
hydrocolloid particles. For example, this can be achieved by the
addition of an inactive form of the ion that will cause
crosslinking of the alginate, which is then activated by a change
in e.g. pH after sufficient dispersion of the ion is complete (see
Glicksman, 1983a; Hoefler, 2004 which are both incorporated herein
by reference). This approach is particularly useful where rapid
gelation is desired and/or where the diffusion approach may lead to
loss of API by diffusion thereof into the crosslinking bath.
[0302] Where another ionotropic polymer is used than alginate,
suitable analogous processes may be used to those described herein
in relation to alginate.
[0303] Following shape-forming, moulding or beading, the resultant
shapes or forms may be washed then dried if appropriate. In the
case of beads solidified in a solidification fluid, an optional
final step in the method of production described above therefore
comprises removal of the solidified beads from the solidification
fluid. This may be achieved e.g. by collection in a mesh basket
through which the solidification fluid (e.g. medium chain
triglycerides) is drained and the beads retained and is preferably
conducted without delay e.g. as soon as the beads have formed or
within 5, 10, 15, 20, 25 or 30 minutes of their formation. Excess
solidification fluid may then be removed using a centrifuge (or
other apparatus or machine adapted to remove excess fluid) followed
by drying of the beads to remove water or free water and/or removal
of some or all of any additional solvent e.g. ethanol or isopropyl
alcohol used to dissolve or facilitate dissolution of the active
principle in preceding steps optionally followed by washing (e.g.
using ethyl acetate) and a subsequent "drying" step to remove
excess solvent (e.g. ethyl acetate). Isopropyl alcohol is an
example of a solvent which is preferably removed later in
processing to reduce residues in the oil or aqueous phase. Drying
can be achieved by any suitable process known in the art such as
use of a drum drier (e.g. Freund Drum dryer which may be part of
the Spherex equipment train if used) with warm air at between
15.degree. C. and 25.degree. C., preferably around 20.degree. C.
leading to evaporation or entrainment of the water by the air. Use
of gelatin as the polymer matrix (e.g. as principal constituent of
the aqueous immobilisation phase) in most cases requires a drying
step and for beads this is preferably achieved by drying in air as
above described. The resultant composition (the composition of the
invention) is essentially dry as described in more detail
above.
[0304] In terms of the way in which self-assembly structure
dispersion droplets may be formed in the first step of the beading
process described above, variations of the above described method
are possible including introducing droplets into a variety of
solidification fluids.
[0305] In general, the beads may be generated by the application of
surface tension between the liquid dispersion (the mixture of the
aqueous and surfactant phases) and an appropriate solidification
fluid such as, for example, gas or liquid in order to create the
spherical or substantially spherical shape of the ultimate
beads.
[0306] Alternatively, the beads may be produced through ejection or
extrusion of the liquid dispersion through an orifice or nozzle
with a certain diameter and optionally subject to selected
vibrational frequencies and/or gravitational flow. Examples of
machines which may be used are the Freund Spherex, ITAS/Lambo,
Globex or Inotech processing equipment. Operation of the Spherex
machine manufactured by Freund as may be desired to manufacture
beads according to the present invention is described in U.S. Pat.
No. 5,882,680 (Freund), the entire contents of which are
incorporated herein by reference. It is preferred to select a
vibrational frequency in the region of 10-15 RPM although the
ultimate choice (and separately the amplitude of vibration
selected) depends on the viscosity of the dispersion to be beaded.
If the polymer matrix is chosen to solidify at lower temperature,
it may be appropriate to maintain the lines to the orifice/nozzle
at a certain temperature to maintain the fluidity of the
solution.
[0307] It will be appreciated, therefore, that the invention
includes a process for manufacturing a composition of the invention
which comprises: forming an aqueous premix which comprises water
and water soluble/dispersible materials (including therefore a
hydrogel-forming polymer) and a surfactant premix which comprises
surfactant, microorganisms and optionally adjuvant and surfactant
soluble/dispersible materials, and combining the two premixes to
form a dispersion (disperse phase) within an aqueous phase
comprising the hydrogel-forming polymer. The dispersion may then be
formed into a shaped unit, for example a bead. More particularly
the manufacture of the composition may optionally comprise:
[0308] (i) forming an aqueous phase premix comprising, or usually
consisting of, a solution in water of water-soluble constituents
(e.g. hydrogel-forming polymer, any water-soluble excipient(s), any
hydrophilic nutrient(s) as described elsewhere herein);
[0309] (ii) forming a surfactant phase premix comprising a mixture
in a surfactant of microorganisms, optionally adjuvant and
optionally other constituents selected from hydrophobic and
amphiphilic constituents (e.g. nutrient(s) as described elsewhere
herein);
[0310] (iii) mixing the two phases to form a dispersion; and
optionally
[0311] (iv) formulating the dispersion into a bead, e.g. ejecting
it through a single orifice nozzle to form droplets which are
caused or allowed to fall into a water immiscible cooling liquid in
which the droplets cool to form beads, and then separating the
beads from the cooling liquid.
[0312] Some manufacturing processes comprise steps (A) to (D) below
or, alternatively, a manufacturing process may comprise a single
one or any combination of steps (A) to (D).
[0313] (A) Exemplary Preparation of Aqueous Phase:
Aqueous phase components are added to water, e.g. purified water,
under agitation e.g. sonication or stirring. The temperature is
gradually increased, for example to 55-75.degree. C. and in
particular 65.degree. C., to achieve complete dissolution of the
solids. The aqueous phase components include a hydrogel forming
polymer, e.g. gelatin or agar and optionally one or more other
excipients, for example D-sorbitol (a plasticiser) and optionally
one or more active ingredients. Possible aqueous phase components
are described elsewhere herein. The aqueous phase components may
comprise microorganism cells (whether intact and/or fragmented).
There are hereby disclosed processes and their products as
described herein in which at least a portion of the microorganism
content, and optionally the whole of the microorganism content, is
provided in the surfactant phase during manufacture. Nonetheless,
the invention does encompass compositions and processes in which
the entire microorganism content is in the aqueous phase.
[0314] The gelatin may be Type A gelatin. In some less preferred
implementations, the gelatin is Type B. The gelatin may have a
Bloom strength of 125-300, optionally of 200-300, for example of
250-300, and in particular 275. The components of the aqueous phase
may be agitated for a period of, for example, from 1 hour to 12
hours to complete preparation of the aqueous phase (aqueous
premix).
[0315] (B) Exemplary Preparation of Surfactant Phase:
Surfactant phase components are added to the surfactant under
agitation e.g. sonication or stirring. The temperature is gradually
increased, for example in the case of the a waxy surfactant such as
Kolliphor HS 15 to, usually, 35-50.degree. C. and in particular
40.degree. C., to achieve complete dissolution of the solids. The
components of the surfactant phase are therefore usually agitated
e.g. stirred until a clear solution is obtained. The components of
the surfactant phase include the surfactant, for example
Kolliphor.RTM. HS15, and optionally one or more active ingredients.
Possible surfactant phase components are described elsewhere
herein. In particular, the surfactant phase may include
microorganism cells (whether intact and/or fragmented) and usually
adjuvant. The components of the surfactant phase may be agitated
for a period of, for example, from 10 hour to 3 hours to complete
preparation of the surfactant phase (surfactant premix).
[0316] At least one of the aqueous phase and the surfactant phase
includes at least one active ingredient.
[0317] (C) Exemplary Mixing of the Two Phases
The aqueous phase and the surfactant phase are mixed. The two
phases may be mixed in a desired weight; for example, the weight
ratio of surfactant phase to aqueous phase may be from 1:1 to 1:10,
e.g. from 1:1 to 1:6 and optionally from 1:1 to 1:4 and in some
cases from 1:3 to 1:4. In other embodiments the weight ratio of
surfactant phase to aqueous phase may be from 1:1 to 1:3, for
example 1:1 to 1:2.5 or from 1:1 to 1:2 such as 1:1.4-1.6. The
resulting solution is agitated, e.g. sonicated or stirred, at an
elevated temperature, e.g. in the case of the surfactant being a
macrogol-15-hydroxystearate, for example Kolliphor HS 15, or having
a melting point similar to that of Kolliphor HS 15 at a temperature
of 55-75.degree. C. and in particular 65.degree. C., to achieve a
homogeneous micelle dispersion, then the homogenous dispersion is
formed into beads. In particular, the homogenous dispersion is
ejected through a single orifice nozzle to form droplets which fall
into a cooling medium. The nozzle is suitably vibrated to
facilitate droplet formation. The nozzle may be vibrated at a
frequency of 2-200 Hz and optionally 15-50 Hz.
[0318] The cooling medium may for example be air or an oil; the oil
is suitably physiologically acceptable as, for example, in the case
of medium chain triglycerides e.g. Miglyol 810N. The cooling medium
may be at a cooling temperature often of less than 15.degree. C.,
for example of less than 10.degree. C. but above 0.degree. C. In
some embodiments the cooling temperature is 8-10.degree. C. The
nozzle size (diameter) is typically from 0.5 to 7.5 mm, e.g. from
0.5 to 5 mm and optionally from 0.5 to 4 mm. In some embodiments,
the nozzle diameter is from 1 to 5 mm for example from 2 to 5 mm,
and optionally from 3 to 4 mm, and in particular may be 3.4 mm.
[0319] The flow rate through a 3.4 mm nozzle is 5 to 35 g/min and
optionally 10 to 20 g/min and for nozzles of different sizes may be
adjusted suitably for the nozzle area.
[0320] In particular embodiments of the processes described above
and elsewhere in the description, mixing of the surfactant phase
and the aqueous phase results in the formation of a microemulsion
or a composition having the characteristics of a microemulsion,
wherein self-assembly structures comprising the surfactant are
dispersed in the aqueous phase comprising the hydrogel forming
polymer as hereinbefore described to provide a clear
thermodynamically stable composition. For example, mixing of the
two phases may result in a microemulsion comprising micelles
comprising a macrogol-15-hydroxystearate, such as for example
Kolliphor HS 15 dispersed in the aqueous phase comprising the
hydrogel-forming polymer such as gelatin.
[0321] (D) Exemplary Processing of Beads
Cooled beads are recovered, for example they may be recovered from
cooling oil after a residence time of 15-60 minutes, for example
after approximately 30 minutes. Beads recovered from a cooling
liquid (e.g. oil) may be centrifuged to eliminate excess cooling
liquid, and then dried. Suitably, drying is carried out at room
temperature, for example from 15-25.degree. C. and optionally from
20-25.degree. C. The drying may be performed in a drum drier, for
example for a period from 6 to 24 hours, e.g. of about 12 hours in
the case of beads dried at room temperature. The dried beads may be
washed, suitably with a volatile non-aqueous liquid at least
partially miscible with water, e.g. they may be washed with ethyl
acetate. The washed beads may be dried at room temperature, for
example from 15-25.degree. C. and optionally from 20-25.degree. C.
The drying may be performed in a drum drier, for example for a
period from 6 to 48 hours, e.g. of about 24 hours in the case of
beads dried at room temperature. Following drying, the beads are
passed through a 1 to 10 mm, optionally 2 to 5 mm to remove
oversized beads and then through a sieve with a pore size of 0.5 to
9 mm optionally 1 to 4 mm to remove undersized beads.
[0322] It can be appreciated that it is possible to recycle the
beads that are rejected by the sieving process.
[0323] The Spherex machine (and others) may be adapted to make use
of a dual concentric lumen nozzle to ensure simultaneous extrusion
of two fluids, the fluid in the inner lumen forming a core and the
fluid of the outer lumen forming a capsule. The fluid forming the
capsule is solidified according to one of the methods described. It
may or may not be desirable for the fluid forming the core to be
susceptible of solidification to yield a particular embodiment of
the composition of the invention.
[0324] The above machinery adapted in this way can be used to
manufacture the composition of the invention in the form of a
capsule in which the core of the composition is filled with a fluid
(a gas or a liquid) as described in the section above entitled
"Shape, Size and Geometry" (noting that the core, like the capsular
material, may be a composition, albeit optionally a distinct
composition, according to the invention i.e. susceptible of
solidification according to one of the methods described above). A
three-lumen nozzle and appropriate tubing may be employed if it is
desired to include an intermediate internal layer e.g. internal
film layer of non-aqueous material on the inner face of the sphere
with the intermediate layer conveniently being solid at room
temperature. Thus, in terms of the softness/hardness of successive
layers, the composition may for example be described as solid:solid
in the case of two layers or solid:solid:solid in the case of 3
layers or liquid/semi-liquid:solid:solid in the case of 3
layers.
[0325] As a further aspect of the invention there is provided a
composition obtainable by (having the characteristic of) any of the
processes described herein.
[0326] The preceding paragraphs describe the formation of uncoated
beads. It is a preferred embodiment of the present invention to
have coated beads which are described in more detail elsewhere
herein. Such coatings may be single or multiple and may be applied
in a number of ways (see separate section).
[0327] With regard to one of the methods described above (ejection
of emulsion through an optionally vibrating nozzle) with two
concentric orifices (centre and outer), the outer fluid may form a
coat (outside the bead) of e.g. polymeric material (polymeric
coating) which may contain an active principle or may impart
controlled release characteristics to the bead and the inner layer
(core) may be a composition according to the invention. The Spherex
machine manufactured by Freund (see U.S. Pat. No. 5,882,680 to
Freund) is preferably used (the entire contents of this patent is
incorporated herein by reference). Other similar ejection or
extrusion apparatus may also be used, for example the ejection
apparatus described hereinbefore.
[0328] Use of the Spherex machine achieves very high
monodispersity. For example, in a typical 100 g, batch 97 g of
beads were between 1.4 to 2 mm diameter or between 1 and 2 mm.
Desired size ranges can be achieved by methods known in the art for
rejecting/screening different sized particles. For example, it is
possible to reject/screen out the larger/smaller beads by passing a
batch first through e.g. a 2 mm mesh and subsequently through a 1.4
mm mesh.
[0329] The 1.4 to 2 mm diameter range is a good size if it is
desired to coat the beads (if smaller, the spray of the coating
machine may bypass the bead; if too large, the beads may be harder
to fluidise which is necessary to achieve consistent coating).
[0330] The beads are preferably internally (i.e. cross-sectionally)
homogeneous i.e. monolithic although processing conditions may be
varied for example by altering the temperature of the fluid
emulsion, the solidification fluid and the concentration of
components in these fluids and the time allowed for certain
processing steps to occur including drying. Although not currently
preferred, such variations may be applied in the case of bead
manufacture to achieve heterogeneity such as, for example, a harder
skin and softer core with less than complete immobilization of oil
droplets towards the core as opposed to the surface of the bead.
Larger (e.g. non-beaded) forms or shapes of the composition
according to the invention may particularly be engineered to embody
such heterogeneity. However, it is currently preferred to have
internally homogenous compositions according to the invention and
within the bead embodiment, this can be favoured by conducting the
beading/dropletting using a homogeneous medium e.g. well dispersed
micelles. Such homogeneity in the micelle dispersion to be beaded
can help avoid the drying conditions affecting symmetry.
[0331] The invention further provides a product having the
characteristics of a composition obtained as described herein, a
product defined in terms of its characteristics being defined by
the characteristics of the composition to the exclusion of the
method by which it was made.
Coating
[0332] A coating may be applied to the beads for targeted,
controlled and/or sustained release of the active(s), in particular
of the microorganism and optional adjuvant. Application of the
appropriate coat may, for example if colonic release is required,
allow for say less than 10% of the active principle to be dissolved
(in dissolution medium) at 4 hours and then a burst (sudden
release) towards a maximum dissolution (approaching 100%) in the
subsequent 24 hours. Many alternative target profiles are possible
and this example is purely for illustration.
[0333] Thus according to one embodiment of the present invention,
there is provided a dosage form comprising a population of beads,
at least some of which and optionally all of which bear a coat
(i.e. are coated) in order to control release of active principle
(microorganism and optional adjuvant) from the bead. In one
embodiment, the coat is a film and, in another embodiment, it is a
membrane. The coat, e.g. film or membrane, may serve to delay
release until after the stomach and to protect the microorganism
and any adjuvant from gastric fluid; the coat may therefore be an
enteric coat. The coat may comprise one or more substances
preferably of a polymeric nature (e.g. methacrylates etc;
polysaccharides etc as described in more detail below) or
combination of more than one such substance, optionally including
other excipients or active principles, such as, for example,
plasticizers, described e.g. in the sections above on active
principles. Preferred plasticizers, if they are used, include
hydrophilic plasticizers for example triethyl citrate (TEC) which
is particularly preferred when using the Eudragit.RTM. family of
polymers as coatings as described below. Another preferred
plasticiser, described in more detail below in relation to coating
with ethyl cellulose, is DBS. Alternative or additional optionally
included excipients are glidants. A glidant is a substance that is
added to a powder or other medium to improve its flowability. A
typical glidant is talc which is preferred when using the
Eudragit.RTM. family of polymers as coatings.
[0334] There may be one or more coatings which comprise active
ingredient(s), for example a nutrient (e.g. combination of
nutrients) which stimulate the immune system as described elsewhere
herein. Such a coating may be an immediate release coating, a
delayed release coating or a sustained release coating. An
immediate release coating comprising such nutrients and/or other
actives may be provided over and/or under a controlled release
coating, for example over and/or under an enteric coating or an
erodible coating.
[0335] In embodiments of the invention the composition comprises a
hydrogel-forming polymer and further polymers able to achieve a
desired delay (or other change) in the release of the drug and/or
poration of the coating and/or exposure of the composition within
the coating to allow egress of drug and/or dissolution of the
immobilization matrix. In one embodiment, the composition comprises
two types of polymers, which are combined into the same polymeric
material, or provided as separate coats that are applied to the
composition.
[0336] Controlled release can be achieved without an additional
coating. In this case the polymer matrix comprises a further
polymer aimed at a controlled release of an active ingredient.
While mixtures of hydrogel-forming polymers are contemplated by the
invention, the composition of the present invention in many
embodiments comprises a polymer matrix material which is
substantially a single material or type of material among those
described herein and/or a matrix which can be solidified without
inclusion of specific additional polymeric components in the
aqueous phase. However, mixtures may be preferred to achieve
certain performance characteristics. Thus it may be desired to
incorporate certain constraining or retarding substances
(retardants) into the water-soluble polymer matrix. In certain
embodiments, such incorporation permits a coat (or coating) to be
dispensed with. In other embodiments where a constraining or
retarding agent is included into the water-soluble polymer matrix,
a coat (or coating) may be present and desirable. For example,
incorporation of a retarding agent which is insoluble in acid
milieu (such as the stomach) is selected to prevent or retard
release in the stomach and a coating may not be needed i.e. the
composition may be free of a coat/coating. Alternatively,
incorporation of a retarding agent which is soluble in acid media
may be selected to retard release in the intestine distal to the
stomach. Again a coating may not be needed i.e. the composition may
be free of a coat/coating. However, the composition according to
the invention which incorporates a retarding agent soluble in acid
media may optionally be coated e.g. with an acid-resistant polymer
to achieve particular advantage. Such a composition is protected
from (complete) gastric release (or gastric release is retarded)
owing to the effect of the acid-resistant polymer coat. Distal to
the stomach, following loss of the coat, the acid-soluble agent
retards release because the milieu of the small and large intestine
is no longer acid.
[0337] Retarding or constraining agents insoluble in acid milieu
include polymers whose solubility is pH-dependent i.e. soluble at
higher pH. Such polymers are described in detail in the section
below entitled "Coating" and such polymers may be used either as
coats/coatings or as retarding agents incorporated into the
water-soluble polymer matrix. An example of a suitable retarding
agent mentioned in the section below entitled "Coating" is HPMCP
(hydroxy-propyl-methyl-cellulose-phthalate also known as
hypromellose phthalate) which is used to prevent release in the
gastric environment since it is soluble above pH 5.5--see that
section for other examples of polymers soluble in non-acid (basic)
media. HPMCP may also be used as a pore-former. Retarding or
constraining agents soluble in acid milieu include polymers whose
solubility is pH-dependent i.e. soluble at lower pH. Such polymers
include cationic polymers such as for example copolymers based on
dimethylaminoethyl methacrylate, butyl methacrylate, and methyl
methacrylate. An example of such a cationic co-polymer which may be
used according to the invention is Eudragit E PO commercially
available from Evonik Industries.
[0338] It has previously been stated that the dosage form of the
invention may comprise more than one population of beads. Within
the coating embodiment, the differences between populations may lie
in the coat i.e. two (or more) populations of beads may differ in a
number of respects one of which is the coating.
[0339] The coat may be applied as described below and may vary as
to thickness and density. The amount of coat is defined by the
additional weight added to (gained by) the dry composition (e.g.
bead) of the invention. Weight gain is preferably in the range 0.1%
to 50%, preferably from 1% to 15% of the dry weight of the bead,
more preferably in the range 3% to 10% or in the range 5-12% or in
the range 8-12%.
[0340] The polymeric coating material may comprise methacrylic acid
co-polymers, ammonio methacrylate co-polymers, or mixtures thereof.
Methacrylic acid co-polymers such as, for example, EUDRAGIT.TM. S
and EUDRAGIT.TM. L (Evonik) are particularly suitable. These
polymers are gastroresistant and enterosoluble polymers. Their
polymer films are insoluble in pure water and diluted acids. They
may dissolve at higher pHs, depending on their content of
carboxylic acid. EUDRAGIT.TM. S and EUDRAGIT.TM. L can be used as
single components in the polymer coating or in combination in any
ratio. By using a combination of the polymers, the polymeric
material can exhibit solubility at a variety of pH levels, e.g.
between the pHs at which EUDRAGIT.TM. L and EUDRAGIT.TM. S are
separately soluble. In particular, the coating may be an enteric
coating comprising one or more co-polymers described in this
paragraph. A particular coating material to be mentioned is
Eudragit L 30 D-55.
[0341] The trademark "EUDRAGIT" is used hereinafter to refer to
methacrylic acid copolymers, in particular those sold under the
EUDRAGIT.TM. by Evonik.
[0342] The coating can comprise a polymeric material comprising a
major proportion (e.g., greater than 50% of the total polymeric
coating content) of at least one pharmaceutically acceptable
water-soluble polymer, and optionally a minor proportion (e.g.,
less than 50% of the total polymeric content) of at least one
pharmaceutically acceptable water insoluble polymer. Alternatively,
the membrane coating can comprise a polymeric material comprising a
major proportion (e.g., greater than 50% of the total polymeric
content) of at least one pharmaceutically acceptable water
insoluble polymer, and optionally a minor proportion (e.g., less
than 50% of the total polymeric content) of at least one
pharmaceutically acceptable water-soluble polymer.
[0343] Ammonio methacrylate co-polymers such as, for example,
EUDRAGIT.TM. RS and EUDRAGIT.TM. RL (Evonik) are suitable for use
in the present invention. These polymers are insoluble in pure
water, dilute acids, buffer solutions, and/or digestive fluids over
the entire physiological pH range. The polymers swell in water and
digestive fluids independently of pH. In the swollen state, they
are then permeable to water and dissolved active agents. The
permeability of the polymers depends on the ratio of ethylacrylate
(EA), methyl methacrylate (MMA), and trimethylammonioethyl
methacrylate chloride (TAMCI) groups in the polymer. For example,
those polymers having EA:MMA:TAMCI ratios of 1:2:0.2 (EUDRAGIT.TM.
RL) are more permeable than those with ratios of 1:2:0.1
(EUDRAGIT.TM. RS). Polymers of EUDRAGIT.TM. RL are insoluble
polymers of high permeability. Polymers of EUDRAGIT.TM. RS are
insoluble films of low permeability. A diffusion-controlled
pH-independent polymer in this family is RS 30 D which is a
copolymer of ethyl acrylate, methyl methacrylate and a low content
of methacrylic acid ester with quaternary ammonium groups present
as salts to make the polymer permeable. RS 30 D is available as an
aqueous dispersion.
[0344] The amino methacrylate co-polymers can be combined in any
desired ratio, and the ratio can be modified to modify the rate of
drug release. For example, a ratio of EUDRAGIT.TM. RS:EUDRAGIT.TM.
RL of 90:10 can be used. Alternatively, the ratio of EUDRAGIT.TM.
RS:EUDRAGIT.TM. RL can be about 100:0 to about 80:20, or about
100:0 to about 90:10, or any ratio in between. In such
formulations, the less permeable polymer EUDRAGIT.TM. RS generally
comprises the majority of the polymeric material with the more
soluble RL, when it dissolves, permitting gaps to be formed through
which solutes can come into contact with the bead allowing
pre-dissolved pharmaceutical actives to escape in a controlled
manner.
[0345] The amino methacrylate co-polymers can be combined with the
methacrylic acid co-polymers within the polymeric material in order
to achieve the desired delay in the release of the drug and/or
poration of the coating and/or exposure of the composition within
the coating to allow egress of drug and/or dissolution of the
immobilization or water-soluble polymer matrix. Ratios of ammonio
methacrylate co-polymer (e.g., EUDRAGIT.TM. RS) to methacrylic acid
co-polymer in the range of about 99:1 to about 20:80 can be used.
The two types of polymers can also be combined into the same
polymeric material, or provided as separate coats that are applied
to the beads.
[0346] Eudragit.TM. FS 30 D is an anionic aqueous-based acrylic
polymeric dispersion consisting of methacrylic acid, methyl
acrylate, and methyl methacrylate and is pH sensitive. This polymer
contains fewer carboxyl groups and thus dissolves at a higher pH
(>6.5). The advantage of such a system is that it can be easily
manufactured on a large scale in a reasonable processing time using
conventional powder layering and fluidized bed coating techniques.
A further example is EUDRAGIT.RTM. L 30D-55 which is an aqueous
dispersion of anionic polymers with methacrylic acid as a
functional group. It is available as a 30% aqueous dispersion.
[0347] In addition to the EUDRAGIT.TM. polymers described above, a
number of other such copolymers can be used to control drug
release. These include methacrylate ester co-polymers such as, for
example, the EUDRAGIT.TM. NE and EUDRAGIT.TM. NM ranges. Further
information on the EUDRAGIT.TM. polymers can be found in "Chemistry
and Application Properties of Polymethacrylate Coating Systems," in
Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms, ed.
James McGinity, Marcel Dekker Inc., New York, pg 109-114 the
entirety of which is incorporated herein by reference.
[0348] Several derivatives of hydroxypropyl methylcellulose (HPMC)
also exhibit pH dependent solubility and may be used in the
invention for coating. These include hydroxypropyl methylcellulose
phthalate (HPMCP), which rapidly dissolves in the upper intestinal
tract and hydroxypropyl methylcellulose acetate succinate (HPMCAS)
in which the presence of ionisable carboxyl groups causes the
polymer to solubilize at high pH (>5.5 for the LF grade and
>6.8 for the HF grade). These polymers are commercially
available from Shin-Etsu Chemical Co. Ltd. As with other polymers
described herein as useful for coatings, HPMC and derivatives may
be combined with other polymers e.g. EUDRAGIT RL-30 D.
[0349] There may be used a polymeric coating substance which is
pH-independent in its dissolution profile and/or in its ability to
release active principles incorporated in the compositions of the
invention. Examples have already been given (e.g., Eudragit RS and
RL). Another example of a pH-independent polymeric coating
substance is ethylcellulose. It will be understood that an
ethylcellulose composition for use in coating a dosage form for may
comprise, in addition to ethylcellulose and--in the case of a
liquid composition--a liquid vehicle, one or more other components.
The other components may serve to modulate the properties of the
composition, e.g. stability. The ethylcellulose may be the sole
controlled release polymer in such a composition. The
ethylcellulose may be in an amount of at least 50%, at least 60%,
at least 70%, at least 80%, at least 90% or at least 95% by weight
of the dry weight of composition for use in coating a dosage form.
Accordingly, an ethylcellulose coating may include other components
in addition to the ethylcellulose. The ethylcellulose may be in an
amount of at least 50%, at least 60%, at least 70%, at least 80%,
at least 90% or at least 95% by weight of the ethylcellulose
coating.
[0350] A particular ethylcellulose coating composition which may be
applied to the compositions of the invention is a dispersion of
ethylcellulose in a sub-micron to micron particle size range, e.g.
from about 0.1 to 10 microns in size, homogeneously suspended in
water with the aid of an emulsification agent, e.g. ammonium
oleate. The ethylcellulose dispersion may optionally and preferably
contain a plasticizer, for example dibutyl sebacate (DBS) or medium
chain triglycerides. Such ethylcellulose dispersions may, for
example, be manufactured according to U.S. Pat. No. 4,502,888,
which is incorporated herein by reference. One such ethylcellulose
dispersion suitable for use in the present invention and available
commercially is marketed under the trademark Surelease.RTM., by
Colorcon of West Point, Pa. USA. In this marketed product, the
ethylcellulose particles are, e.g., blended with oleic acid and a
plasticizer, then optionally extruded and melted. The molten
plasticized ethylcellulose is then directly emulsified, for example
in ammoniated water optionally in a high shear mixing device, e.g.
under pressure. Ammonium oleate can be formed in situ, for instance
to stabilize and form the dispersion of plasticized ethylcellulose
particles. Additional purified water can then be added to achieve
the final solids content. See also U.S. Pat. No. 4,123,403, which
is incorporated herein by reference.
[0351] The trademark "Surelease.RTM." is used hereinafter to refer
to ethylcellulose coating materials, for example a dispersion of
ethylcellulose in a sub-micron to micron particle size range, e.g.
from about 0.1 to 10 microns in size, homogeneously suspended in
water with the aid of an emulsification agent, e.g. ammonium
oleate. In particular, the trademark "Surelease.RTM." is used
herein to refer to the product marketed by Colorcon under the
Surelease.RTM. trademark.
[0352] Surelease.RTM. dispersion is an example of a combination of
film-forming polymer, plasticizer and stabilizers which may be used
as a coating to adjust rates of active principle release with
reproducible profiles that are relatively insensitive to pH. The
principal means of drug release is by diffusion through the
Surelease.RTM. dispersion membrane and is directly controlled by
film thickness. Use of Surelease.RTM. is particularly preferred and
it is possible to increase or decrease the quantity of
Surelease.RTM. applied as coating in order to modify the
dissolution of the coated composition. Unless otherwise stipulated,
use of the term "Surelease" may apply to Surelease E-7-19020,
E-7-19030, E-7-19040 or E-7-19050. E-7-19020 comprises
ethylcellulose blended with oleic acid and dibutyl sebacate, then
extruded and melted. The molten plasticized ethylcellulose is then
directly emulsified in ammoniated water in a high shear mixing
device under pressure. Ammonium oleate is formed in situ to
stabilize and form the dispersion of plasticized ethylcellulose
particles. Additional purified water is then added to achieve the
final solids content. E-7-19030 additionally comprises colloidal
anhydrous silica dispersed into the material. E-7-19040 is like
E-7-19020 except that it comprises medium chain triglycerides
instead of dibutyl sebacate. E-7-19050 derives from blending
ethylcellulose with oleic acid before melting and extrusion. The
molten plasticized ethylcellulose is then directly emulsified in
ammoniated water in a high shear mixing device under pressure.
Ammonium oleate is formed in situ to stabilize and form the
dispersion of plasticized ethylcellulose particles. However,
E-7-19040 is preferred.
[0353] The invention also contemplates using combinations of
Surelease with other coating components, for example sodium
alginate, e.g. sodium alginate available under the trade name
Nutrateric.TM..
[0354] In addition to the EUDRAGIT.TM. and Surelease.RTM. polymers
discussed above, other polymers may be used, in particular enteric,
or pH-dependent, polymers. Such polymers can include phthalate,
butyrate, succinate, and/or mellitate groups. Such polymers
include, but are not limited to, cellulose acetate phthalate,
cellulose acetate succinate, cellulose hydrogen phthalate,
cellulose acetate trimellitate, hydroxypropyl-methylcellulose
phthalate, hydroxypropylmethylcellulose acetate succinate, starch
acetate phthalate, amylose acetate phthalate, polyvinyl acetate
phthalate, and polyvinyl butyrate phthalate. Additionally, where
compatible, any combination of polymer may be blended to provide
additional controlled- or targeted-release profiles.
[0355] The coating can further comprise at least one soluble
excipient to increase the permeability of the polymeric material.
Suitably, the at least one soluble excipient is selected from among
a soluble polymer, a surfactant, an alkali metal salt, an organic
acid, a sugar, and a sugar alcohol. Such soluble excipients
include, but are not limited to, polyvinyl pyrrolidone,
polyethylene glycol, sodium chloride, surfactants such as, for
example, sodium lauryl sulfate and polysorbates, organic acids such
as, for example, acetic acid, adipic acid, citric acid, fumaric
acid, glutaric acid, malic acid, succinic acid, and tartaric acid,
sugars such as, for example, dextrose, fructose, glucose, lactose,
and sucrose, sugar alcohols such as, for example, lactitol,
maltitol, mannitol, sorbitol, and xylitol, xanthan gum, dextrins,
and maltodextrins. In some embodiments, polyvinyl pyrrolidone,
mannitol, and/or polyethylene glycol can be used as soluble
excipients. The at least one soluble excipient can be used in an
amount ranging from about 1% to about 10% by weight, based on the
total dry weight of the polymer.
[0356] The modifications in the rates of release, such as to create
a delay or extension in release, can be achieved in any number of
ways. Mechanisms can be dependent or independent of local pH in the
intestine, and can also rely on local enzymatic activity to achieve
the desired effect. Examples of modified-release formulations are
known in the art and are described, for example, in U.S. Pat. Nos.
3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533;
5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556;
and 5,733,566 all of which are incorporated herein by reference in
their entirety.
[0357] The addition to Surelease.TM. or other pH-independent
polymer substance of a second polymer (e.g. a polysaccharide,
especially a heteropolysaccharide) which is susceptible to
degradation by colonic bacterial enzymes (and optionally or
alternatively by pancreatic or other relevant enzymes), provides
targeted release of actives to a site or sites where the second
polymer is degraded and flexibility in modulating the amount of
polymer added to the composition of the invention in order to
achieve optimal dissolution profiles.
[0358] The invention therefore also provides a coating for
compositions (whether of the invention or not) intended to release
their active payload in the colon which is a combination of
ethylcellulose (preferably formulated with an emulsification agent
such as, for example, ammonium oleate and/or a plasticizer such as,
for example, dibutyl sebacate or medium chain triglycerides) and a
polysaccharide susceptible of degradation by a bacterial enzyme
normally found in the colon. Such polysaccharides include
chondroitin sulphate, pectin, dextran, guar gum and amylase,
chitosan etc and derivatives of any of the foregoing. Chitosan is
particularly preferred in connection with obtaining a
colon-specific release profile. The invention also includes a
composition comprising a combination of ethylcellulose (preferably
formulated with an emulsification agent such as, for example,
ammonium oleate and/or a plasticizer such as, for example, dibutyl
sebacate or medium chain triglycerides) and a polysaccharide
susceptible of degradation by a bacterial enzyme normally found in
the colon; the composition may include a liquid vehicle, e.g.
water.
[0359] The use of polysaccharides by themselves for coating
purposes has been tried with limited success. Most of the
non-starch polysaccharides suffer from the drawback of lacking good
film forming properties. Also, they tend to swell in the GI tract
and become porous, resulting in the early release of the drug. Even
amorphous amylose, which is resistant to degradation by pancreatic
alpha amylase but capable of degradation by colonic bacterial
enzymes has the disadvantage of swelling in aqueous media although
this can be controlled by incorporating insoluble polymers like,
ethyl cellulose and acrylates into the amylose film. Amylose
however is not water-soluble and although water-soluble
polysaccharides are not excluded, the present inventors have found
that use of a water-soluble polysaccharide (WSP) susceptible of
bacterial enzymic degradation brings particularly advantageous
results when used as a coating in accordance with this embodiment
of the present invention. A particularly preferred polysaccharide
in this embodiment of the present invention is pectin. Various
kinds of pectin may be used including pectin of different grades
available i.e. with differing degrees of methylation (DM), i.e.
percentage of carbonyl groups esterified with methanol, for example
pectins with a DM of more than 50%, known as High Methoxy (HM)
Pectins or Low Methoxy (LM) pectins, or a pectin combination
comprising an HM pectin and an LM pectin. It is also possible in
this embodiment to use pectins having various degrees of
acetylation (DAc). Taken together, the DM and DAc or the degree of
substitution is known as Degree of Esterification (DE). Pectins of
various DE's may be used according to the invention. As an
alternative to pectin, sodium alginate may be used as a
polysaccharide according to an embodiment of the invention.
However, other embodiments may conveniently include amylose and/or
starch which contains amylose. Various grades of starch, containing
different percentages of amylose may be used including for example
Hylon V (National Starch Food Innovation) which has an amylose
percentage of 56% or Hylon VII which has an amylose percentage of
70%. The remaining percentage is amylopectin. The polysaccharides
pectin, amylose and sodium alginate are particularly preferred for
achieving colon delivery i.e. for compositions intended to release
active principles in the colon.
[0360] It has been found that pectin can act as a former of pores
in the coating otherwise provided by ethylcellulose (preferably
Surelease). By "pores" is not meant shaft-like holes from the
surface to the core of the composition, rather areas of weakness or
absence of coating occurring stochastically on and within the
coating of the invention.
[0361] Pore formers have been described before in connection with
Surelease (see e.g. US 2005/0220878) but in relation to
"gastro-insoluble" substances such as, for example, alginate.
[0362] According to a particular embodiment of the invention, where
the water-soluble polysaccharide (WSP) is pectin, the proportion of
Surelease.TM. to pectin is ideally in the range 90:10 to 99:1,
preferably, 95:5 to 99:1, more preferably 98:2 to 99:1.
[0363] In this particularly preferred combination (Surelease.TM.
WSP e.g. pectin) the weight gain and ratio between Surelease.TM.
and WSP can be varied to refine the behaviour of the coating and
the composition of the invention when it bears such a coat. Thus to
the inventors/applicant's surprise, the advantages of this
preferred combination of coating polymers were further pronounced
by selecting a weight gain in the range 0 to 30% (preferably 5 to
10%) and a Surelease to pectin ratio in the range 95:5 to 99.5:0.5
preferably 97:3 to 99:1 inclusive. Particularly favoured weight
gains using Surelease are those in the range 5-12% or in the range
8-12%.
[0364] Although the focus above has been on extending and/or
sustaining release of active principles from compositions according
to the invention, also contemplated are uncoated or simple enteric
coated compositions providing early, small intestinal active
ingredient release with sufficient enteric coating merely to
protect the composition from dissolution in the stomach.
[0365] It is preferred to dry the composition of the invention
before they are coated with a suitable polymeric coat (as described
in more detail above/below). It is also preferred, in certain
embodiments to apply a first coat before applying a second. In
general the first coat and the second coat may be of the same or
different materials and be chosen from any of the classes of
coating material described herein. In specific embodiments, the
first coat optionally protects the core (e.g. bead) from
interaction with the second coat and/or prevents leaching of
composition contents into the second coat. For example, the first
coat may comprise or be hypromellose, e.g. it may be made of a
mixture of hypromellose, titanium dioxide and polyethylene glycol;
the first coat may comprise at least 50 wt % hypromellose and
optionally at least 75 wt % hypromellose, e.g. at least 80 wt % or
at least 85 wt % or 90 wt % hypromellose. The coating material used
to form the first coat may therefore comprise a dry weight
percentage of hypromellose mentioned in the preceding sentence. The
second (outer) coat may be an enteric coating as described above or
comprise a mixture of polymers including a polymer degradable by
bacterial or other enzymes, for example be made of the
Surelease-pectin mixture described above. If it is desired for the
first coat to use a mixture of hypromellose, titanium dioxide and
polyethylene glycol, commercial products corresponding to such
mixtures are available including Opadry White, a product
commercialised by Colorcon. More generally, there may be mentioned
various products commercialised under the trade name Opadry and
Opadry II. Further nonlimiting examples include Opadry YS-1-7706-G
white, Opadry Yellow 03B92357, Opadry Blue 03B90842). These
compositions are available as dry film coating compositions that
can be diluted in water shortly before use. Opadry and Opadry II
formulations comprise a cellulosic film forming polymer (e.g., HPMC
and/or HPC), and may contain polydextrose, maltodextrin, a
plasticizer (e.g., triacetin, polyethylene glycol), polysorbate 80,
a colorant (e.g., titanium dioxide, one or more dyes or lakes),
and/or other suitable film-forming polymers (e.g.,
acrylate-methacrylate copolymers). Suitable OPADRY or OPADRY II
formulations may comprise a plasticizer and one or more of
maltodextrin, and polydextrose (including but not limited to a)
triacetin and polydextrose or maltodextrin or lactose, or b)
polyethylene glycol and polydextrose or maltodextrin). Particularly
preferred commercial products are Opadry White (HPMC/HPC-based) and
Opadry II White (PVA/PEG-based). Alternative (non-Opadry) products
for initial protective coats include polyvinyl alcohol-polyethylene
glycol graft copolymers such as is available commercially under the
name Kollicoat IR and methyl methacrylate ammonium-based copolymers
such as are available commercially under the name Eudragit E.
Another preferred example is low molecular weight HPMC. The
optional inner coat is applied in the same manner as is the outer
(or sole) coat (or coating layer).
[0366] The coating process can be carried out by any suitable means
such as, for example, by use of a coating machine which applies a
solution of a polymer coat (as described above in particular) to
the composition. Polymers for coating are either provided by the
manufacturer in ready-made solutions for direct use or can be made
up before use following manufacturers' instructions.
[0367] Appropriate coating machines are known to persons skilled in
the art and include, for example, a perforated pan or
fluidized-based system for example the GLATT, Vector (e.g. CF 360
EX), ACCELACOTA, Diosna, O'Hara and/or HICOATER processing
equipment. To be mentioned is the MFL/01 Fluid Bed Coater (Freund)
used in the "Bottom Spray" configuration.
[0368] Typical coating conditions are as follows:
TABLE-US-00001 Process Parameter Values Fluidising airflow (m3/h)
20-60 (preferably 30-60) Inlet air temperature (.degree. C.) 20-65
Exhaust air temperature (.degree. C.) 20-42 Product temperature
(.degree. C.) 20-42 Atomizing air pressure (bar) Up to 1.4 e.g.
0.8-1.2 Spray rate (g/min) 2-10 and 3-25 RPM
[0369] The compositions of the invention may be coated with active
(nutrient and/or drug) layers using methods conventional in the art
of pharmaceutical science (such as for example using coating
machines as just described) to produce a composition having one or
more layer(s), each layer containing one or more active nutrient,
pharmaceutical or other ingredient/excipient as described elsewhere
herein. Drug layering means the deposition of at least one or
successive layers of drug entities from solution, suspension or dry
powder on nuclei e.g. beads as described herein. The active
ingredient optionally may be free of excipients or in combination
with one or more excipients.
[0370] Drug layering includes solution/suspension layering, powder
layering and powder drug layering. In solution/suspension layering,
drug particles are dissolved or suspended in a binding liquid. In
powder layering, complete dissolution does not occur, due to low
liquid saturation, irrespective of the solubility of the active
agent in the binding liquid. In powder drug layering, a binder
solution is first sprayed onto previously prepared seeds e.g. beads
as described herein, followed by the addition of powder.
Conventional pan coaters may be used as described above for polymer
coating although modified forms of pan coaters are preferred
including fluidised-bed and centrifugal rotary granulators.
Examples of suitable granulators include the Rotor granulator.
(Glatt), the Rotor-processor (Aeromatic), the Spir-a-Flow (Freund)
and the CF-granulator (Freund).
[0371] Further examples of drug layering techniques which may be
applied to compositions of the invention and/or incorporated in
manufacturing methods of the invention are dry coating as described
by Luo et al (International Journal of Pharmaceuticals, 358,
(2008), page 16-22). Luo et al describe a number of dry coating
methods suitable for use in the present invention:
electrostatic-dry coating; plasticiser-dry-coating;
heat-dry-coating; and plasticizer-electrostatic-heat-dry-coating.
Heat-dry-coating uses heat, and the resulting partially melted
surface of the powder particles, as the sole binding force. The
coating process is achieved by spreading the coating material
comprising active ingredient onto the beads in a spheroniser. The
coating materials are spread onto the beads by for example a screw
powered feeder. The coating material is heated by any means known
in the art (e.g. with an infra-red lamp). This technique can be
used with neat coating material or with a coating material pre
dosed with a plasticiser.
[0372] In certain embodiments the drug layering material comprises
an active ingredient, e.g. nutrient, and a surfactant, in
particular a surfactant as described herein as one useful for
forming self-assembly structures and optionally a surfactant the
same as the self-assembly structures-forming surfactant combined
with the hydrogel-forming polymer. The surfactant used in drug
layering is conveniently a waxy surfactant solid at room
temperature (in particular solid at 25.degree. C. and desirably
solid at 30.degree. C.). Such drug layering may be carried out by
the solution/suspension or solid method as detailed above. The drug
layering may be achieved following the techniques described by Luo
et al. in International Journal of Pharmaceuticals, 358, (2008),
page 16-22. The surfactant may be selected from those disclosed
above, therefore, for example it may be a macrogol ester, e.g. of a
fatty acid, and particularly macrogol-15-hydroxystearate, and more
specifically Kolliphor HS 15. In a representative example,
compositions (particularly beads) of the invention are layered with
an active ingredient and macrogol-15-hydroxystearate; the invention
therefore includes compositions having one or more layers which
include at least one layer comprising a surfactant, e.g.
macrogol-15-hydroxystearate, and a drug. In certain embodiments the
macrogol-15-hydroxystearate or other surfactant is layered onto the
beads using the heat-dry-coating technique described above. The
surfactant which is incorporated in said at least one layer may be
the same as, or sometimes different from, the surfactant which
forms the self-assembly structures.
[0373] The use of beads of the invention as seeds for drug layering
is superior to using traditional non-pareils as initial substrates
in the preparation of pellets by a drug layering process. One
reason is the optimal size of the beads of the current invention.
Another reason is that sucrose, the main component of traditional
non-pareils, has well-known drawbacks including harmful effects on
diabetics and potential cariogenicity. According to the prior art,
microcrystalline cellulose (MCC) has also been tested as a
substrate for drug layering although the inventors/applicants are
not aware of successful use of MCC for the preparation of initial
cores/beads in a centrifugal granulating process as may be used in
embodiments of the present invention. Thus in one embodiment, the
invention provides a process for the manufacture of drug-coated
pellets comprising using the beads described herein as seeds or as
non-pareils (i.e. instead of non-pareils) on which the drug is
coated. In a related embodiment, a composition of the invention
comprises a bead of the disclosure coated with one or more drug
layers. Another embodiment is a process of enhancing the solubility
of poorly water-soluble active principles by using one or more of
the above described methods of drug layering, including
spray-drying-based processes. The polymeric coat, described in
detail above, may or may not be applied to a drug-layered bead.
However, if desired, it may be applied after such drug layering. In
applying a drug layer, the drug to be layered onto the bead may
optionally first be admixed with appropriate excipients such as,
for example, binders as described elsewhere herein. A particularly
preferred binder in this context is polyvinyl pyrrolidone (also
spelt polyvinylpyrrolidone and also known as PVP or povidone). PVPs
of various K-values may be used. The K-value of PVP is a function
of its average molecular weight, the degree of polymerization, and
the intrinsic viscosity. It is particularly preferred to use PVP
K-32. Up to 5% of the dry weight of the composition of the
invention in this embodiment may be made up of such binders.
Approximately 1% or less is preferred. Other suitable binders which
may be used in drug-layering include gelatin, carboxymethyl
cellulose, hydroxypropyl methylcellulose and hydrolysed starches
e.g. maltodextrins. Compositions embodying drug layering may also
optionally be coated with a polymer coating, or include a polymer
layer, to control release as described more generally above
including the option to include the same or a different active
principle in this polymer coat.
[0374] The invention therefore includes a layered bead
comprising:
[0375] a core comprising, or consisting of, a hydrogel-forming
polymer matrix material in which are dispersed (i) micelles and/or
pro-micelles, and (ii) an active ingredient comprising an antigen
selected from live, killed, attenuated and inactivated
microorganisms; and
[0376] a layer surrounding the core and comprising an active
ingredient, which may be the same as or different from the active
ingredient comprised in the core, the active ingredient layer
optionally also having controlled release properties or other
functionality.
The core may additionally comprise an adjuvant. Any adjuvant and
the antigen may be included in the micelles and/or promicelles or
be included in the polymer matrix, or be included in both.
[0377] The layered bead may have a plurality of layers, e.g. 2, 3,
4 or 5 layers, comprising an active ingredient, wherein the active
ingredient of each layer is selected independently from the active
ingredient of each other layer. In one embodiment, each layer
comprises the same active ingredient as each other layer; in
another embodiment, no two layers comprise the same active
ingredient. The term "active ingredient" in this paragraph embraces
both a single active entity and a combination of active entities.
The layered bead may comprise one or more polymer layers, to
control release as described more generally above. Such a polymer
layer may contain an active ingredient and therefore constitute a
drug layer as well as a release control layer. Alternatively, a
polymer layer may be free of active ingredient. A polymer layer,
whether or not it contains an active ingredient, may be located
between the core and a drug layer outside the polymer layer, or
between two drug layers, or may form an outer layer.
[0378] The invention therefore includes a layered bead
comprising
[0379] a core comprising, or consisting of, a matrix comprising a
hydrogel-forming polymer; and comprised in the matrix, a
microorganism selected from live, killed, attenuated and
inactivated microorganisms, a surfactant and an adjuvant;
[0380] an active ingredient layer surrounding the core and
comprising an active ingredient, which may be the same as or
different from the active principle comprised in the core, the
active ingredient layer optionally also having controlled release
properties or other functionality; and
[0381] a polymer layer free of active ingredient.
[0382] The polymer layer may be located between the core and the
active principle layer. The polymer layer may be located externally
of the active principle layer. The layered bead may comprise a
plurality of active principle layers and, additionally or
alternatively, it may comprise a plurality of polymer layers. In
some embodiments, there is at least one active principle layer
which comprises a release-controlling polymer. In some embodiments,
the outermost layer comprises a release-controlling polymer, which
may contain an active ingredient or, in another implementation, be
free of active principle.
[0383] The optionally coated beads of the invention may be
formulated directly following their manufacture in the ways
described above. In an alternative embodiment, it may be desired to
impart different properties to the beads and/or to a final dosage
form. One way of achieving this according to the invention is
through granulation e.g. to improve the flow of powder mixtures of
beads with other components as e.g. described above in relation to
binders. Granules of intact or broken beads may be obtained by
adding liquids (e.g. binder or solvent solutions) and effecting a
granulating step as described in the prior art. Larger quantities
of granulating liquid produce a narrower particle size range and
coarser and harder granules, i.e. the proportion of fine granulate
particles decreases. The optimal quantity of liquid needed to get a
given particle size may be chosen in order to minimise
batch-to-batch variations. According to this embodiment, wet
granulation is used to improve flow, compressibility,
bio-availability, homogeneity, electrostatic properties, and
stability of the composition of the invention presented as a solid
dosage form. The particle size of the granulate is determined by
the quantity and feeding rate of granulating liquid. Wet
granulation may be used to improve flow, compressibility,
bio-availability, and homogeneity of low dose blends, electrostatic
properties of powders, and stability of dosage forms. A wet
granulation process according to this embodiment may employ low or
high shear mixing devices in which a low viscosity liquid
(preferably water) is added to a powder blend containing binder
previously dry mixed with the rest of the formulation including
beads. Alternative granulation approaches which may be utilized
include high-shear, extrusion and conventional wet granulation.
Dosage Forms
[0384] In a further aspect, the present invention provides for a
dosage form comprising a population of beads of the invention. The
bead of the dosage form comprises a hydrogel-forming polymer, a
surfactant, and an active ingredient. The beads of the dosage form
may optionally be coated (as described above). In certain
embodiments the dosage form may comprise at least two populations
of beads.
[0385] Where the bead of the dosage form comprises an active
ingredient, which is an active pharmaceutical ingredient the dosage
form is suitable for pharmaceutical use.
[0386] The dosage form is obtainable by preparing a bead comprising
a hydrogel-forming polymer, a surfactant in the form of micelles
dispersed in the polymer and an active ingredient. Optionally, the
bead is coated; the optional coating may be formulated in such a
way as to provide a known or desired release profile in the
gastrointestinal tract (GIT). A population of beads is then
formulated into a suitable single unit dosage form (as described
below) by procedures known to those skilled in the art to produce
the dosage form. The dosage form may be further processed (e.g. by
coating) to allow a modified release rate of the active ingredient
in the GIT.
[0387] In certain embodiments the dosage form comprises a
population of beads of the invention in a unit dosage form suitable
for administration, for example to a human or animal. The unit
dosage form chosen from a capsule, a tablet, a sprinkle, a sachet,
a suppository, a pessary or other suitable unit dosage form.
[0388] In embodiments the dosage form comprising a population of
beads may be presented in a single unit dosage form e.g. contained
in a single hard gel capsule which releases the beads e.g. in the
stomach. Alternatively the beads may be presented in a sachet or
other container which permits the beads to be sprinkled onto food
or into a drink or to be administered via a feeding tube for
example a naso-gastric tube or a duodenal feeding tube.
Alternatively, the beads may be administered as a tablet for
example if a population of beads is compressed into a single tablet
as described below. Alternatively, the beads may be filled e.g.
compressed into a specialist bottle cap or otherwise fill a space
in a specialised bottle cap or other element of a sealed container
(or container to be sealed) such that e.g. on twisting the bottle
cap, the beads are released into a fluid or other contents of the
bottle or vial such that the beads are dispersed (or dissolve) with
or without agitation in such contents. An example is the Smart
Delivery Cap manufactured by Humana Pharma International (HPI)
S.p.A, Milan, Italy.
[0389] The dosage form may be formulated in such a way so that the
beads of the invention can be further developed to create a larger
mass of beads e.g. via compression (with appropriate oil or
powder-based binder and/or filler known to persons skilled in the
art of pharmaceutical formulation and with the option of including
additional quantities of the same API as in the composition of the
invention or a different API a preferred example being where the
composition of the invention takes the form of beads which comprise
immediate or controlled release cyclosporine and the binder or
filler comprises MMF, mycophenolate mofetil, an immunosuppressant)
of a plurality of beads which disintegrate at a different rate in
different conditions than a unitary moulded form of the same shape.
The larger (e.g. compressed) mass may itself take a variety of
shapes including pill shapes, tablet shapes, capsule shapes etc. A
particular problem which this version of the bead embodiment solves
is the "dead space" (above the settled particulate contents) and/or
"void space" (between the particulate content elements) typically
found in hard gel capsules filled with powders or pellets. In such
pellet- or powder-filled capsules with dead/void space, a patient
is required to swallow a larger capsule than would be necessary if
the capsules contained no such dead space. The beads of this
embodiment of the invention may readily be compressed into a
capsule to adopt the inner form of whichever capsule or shell may
be desired leaving much reduced, e.g. essentially no, dead/void
space. Alternatively the dead or void space can be used to
advantage by suspending beads in a vehicle such as, for example, an
oil which may be inert or may have functional properties such as,
for example, permeability enhancement or enhanced dissolution or
may comprise an active ingredient being the same or different from
any active ingredients in the bead. For example, hard gelatin
capsules may be filled with a liquid medium combined with uncoated
and/or coated beads. The liquid medium may be one or more of the
surfactant phase constituents described herein or it may be one or
more surfactants. Particularly preferred but non-limiting examples
are corn oil, sorbitane trioleate (sold under the trade mark SPAN
85), propylene glycol dicaprylocaprate (sold under the trade mark
Labrafac), 2-(2-ethoxyethoxyl)ethanol (sold under the trade mark
Trancutol P) and polysorbate 80 (sold under the trade mark Tween
80). An example of a liquid medium which may be used in this
embodiment and which contains an active principle is the
commercially available cyclosporin pre-microemulstion Neoral.TM..
It is particularly preferred to formulate beads according to the
invention in Neoral and to fill a hard gel capsule.
[0390] The beads so-presented may be of a single type (or
population) or may be of multiple types (or populations) differing
between populations in relation to one or more features described
herein e.g. different active ingredient or different excipients or
different physical geometry, coated, multiply coated, uncoated
etc.
[0391] In a representative embodiment the bead of the dosage form
is formed by preforming a self-assembly structure dispersion by
mixing together at least the following materials: a
hydrogel-forming polymer; a surfactant; and a microorganism
selected from live, killed, attenuated and inactivated
microorganisms and optionally additionally mixing together
therewith an adjuvant. The dispersion is immobilized within the
solidified bead by ejection from a single orifice nozzle into a
suitable cooling liquid. Following removal of the drying liquid and
any optional coating, the bead is filled into a gelatin capsule
suitable for pharmaceutical use.
[0392] In some embodiments the dosage form has been appropriately
formulated in such a way as to release the one or more active
ingredients at a specified point in the GIT e.g. the colon.
[0393] Where the dosage form comprises at least two populations of
beads at least some of the beads (e.g. a first population) may
comprise an active ingredient (or more than one) and other beads
(e.g. a second population) may comprise an active ingredient (or
more than one). At least one population comprises an active
ingredient which comprises an antigen selected from live, killed,
attenuated and inactivated microorganisms and optionally comprises
an adjuvant. One population may be free of active principles or
include "deactivating" principles e.g. enzyme or toxin sequesters
or include active excipients, such as, for example, permeability
enhancers, which may enhance, moderate or potentiate the effect of
an active principle in another population. In related embodiments,
the dosage form of the invention may comprise multiple populations
of beads. The active principles may be the same or different as
between populations. The two populations of beads may, in certain
embodiments, be differentially coated, whether singularly or
multiply, so as to provide different release profiles of the same
or different active ingredient(s).
[0394] The dosage form of the invention is suitable for oral
administration.
[0395] The invention includes oral dosage forms comprising multiple
shaped units, e.g. beads, of the invention, therefore.
[0396] Where compositions include at least two active agents, the
composition may as previously described comprise at least two
agents (one being a microorganism selected from live, killed,
attenuated and inactivated microorganisms) within a
hydrogel-forming polymer bead or shaped unit (whether within the
polymer phase or the surfactant phase, or both) for co-release,
and/or it may comprise at least two agents in different parts of
the composition for sequential, e.g. pulsed, release. From the
aspect of sequential release, the composition may comprise an
active agent within a hydrogel-forming polymer bead or shaped unit
(whether within the polymer phase or the surfactant phase, or both)
and an active agent in a coating layer; optionally it may comprise
two or more active agents in different coating layers or the same
active agent in two or more different coating layers.
Examples
[0397] In the following examples, all percentages and ratios are by
weight.
[0398] The examples describe the preparation of beads of generally
spherical shape and typically having a diameter of 1 to 2 mm. The
beads are manufactured according to the following general
method:
Method of Preparation
Surfactant Phase
[0399] The antigen (i.e. microorganism) and the adjuvant(s) are
dissolved/dispersed in Kolliphor HS 15. When the antigen or the
adjuvant is supplied in an aqueous solution, the solution is mixed
with Kolliphor HS 15 until a homogeneous mixture is achieved. The
temperature is kept at 35-40.degree. C. to maintain the Kolliphor
HS as a liquid.
Gelatin Phase
[0400] D-Sorbitol is dissolved in water at room temperature, then
gelatin is added and the temperature is increased up to
60-70.degree. C. The solution is stirred until complete dissolution
of the components. (The adjuvant aqueous solution can be used to
prepare the gelatin phase if required, optionally in combination
with water, but this option is not a feature of the examples).
Mixing of the Two Phases
[0401] Surfactant Phase and Gelatin phase are mixed at different
w/w ratios (as shown in table 1). The resulting mixture is stirred
at 60-70.degree. C. to achieve homogeneity. The homogeneous
solution is ejected through a single orifice to form droplets which
fall into a cooling oil medium (Miglyol 810N) at 8-10.degree. C.
The nozzle size (diameter) may be from 0.5 to 3.5 mm.
[0402] After approximately 30 minutes, beads are recovered from the
cooling oil solution, centrifuged to eliminate excess oil and then
dried at room temperature.
[0403] All formulations have been coated with Eudragit L 30 D
55.
[0404] In the examples herein the number of cells present in the
final dry beads obtained following manufacture was not determined.
Accordingly the antigen levels quoted in the beads used in the
experiments are target levels assuming that all of the cells used
in the preparation of the beads were present in the final dried
beads. The actual number of cells in the final dry beads used in
the mouse studies may therefore have been lower than the target
level as a result of losses during the preparation of the
beads.
[0405] ETEC Compositions
[0406] Specifically, Formulations 1 and 2 were obtained as
follows:
[0407] Formulation 1.
[0408] Surfactant Premix: Kolliphor HS 15 (18.02%), ETEC
Suspension* (81.98%).
[0409] Aqueous Premix: Gelatin (17.15%), D-Sorbitol (1.68%), ETEC
Suspension*(81.18%). *ETEC suspension contains 1.times.10 10 cells
per gram.
[0410] The two phases are mixed at a weight ratio of 1:1.69
surfactant premix to aqueous premix.
[0411] Dry Composition:
TABLE-US-00002 Component % Kolliphor HS 15 36.21 Gelatin 58.11
D-sorbitol 5.68 ETEC Cells See note below Note: 4.8 .times.
10{circumflex over ( )}10 ETEC cells have been used to manufacture
Formulation 1; given a batch size of 1093.1 mg and an average
weight of 1 bead equal to 2.1 mg, the total number of beads in the
batch is 1093.1 mg/2.1 = 521 beads. The concentration of ETEC cells
per bead is therefore: 4.8 .times. 10{circumflex over ( )}10/521 =
9.2 .times. 10{circumflex over ( )}7 cell per bead.
[0412] Beads were coated to achieve 5.9% weight gain of Eudragit L
30 D-55.
[0413] Formulation 2.
[0414] Surfactant premix: Kolliphor HS 15 (14.81%), alphaGalCer
(0.08%), ETEC suspension* (85.11%).
[0415] Aqueous Premix: Gelatin (17.17%), D-Sorbitol (1.71%), ETEC
Suspension* (81.12%).
[0416] *ETEC suspension contains 1.times.10 10 cells per gram.
[0417] The two phases are mixed at a weight ratio of 1:1.43
surfactant premix to aqueous premix.
[0418] Dry Composition:
TABLE-US-00003 Component % Kolliphor HS 15 35.31 Gelatin 58.66
D-sorbitol 5.85 Alpha-Gal-Cer 0.18 ETEC Cells See note below Note:
4.8 .times. 10{circumflex over ( )}10 ETEC cells have been used to
manufacture Formulation 1; given a batch size of 1084.4 mg and an
average weight of 1 bead equal to 2.1 mg, the total number of beads
in the batch is 1084.4 mg/2.1 = 517 beads. The concentration of
ETEC cells per bead is therefore: 4.8 .times. 10{circumflex over (
)}10/521 = 9.3 .times. 10{circumflex over ( )}7 cell per bead.
[0419] Given the batch size of 1084.4 mg dry weight, the absolute
quantity (dry weight) of each component in the batch was as
follows:
TABLE-US-00004 Component Quantity Kolliphor HS 15 382.90 mg Gelatin
636.11 mg D-sorbitol 3.48 mg Alpha-Gal-Cer 1.95 mg ETEC Cells 4.8
.times. 10{circumflex over ( )}10
[0420] A summary of various ratios of Formulation 2 is presented in
the following table:
TABLE-US-00005 Ratio (dry weight mg to 1 .times. 10{circumflex over
( )}10 cells) Value Kolliphor HS 15: ETEC Cells 79.77 mg: 1 .times.
10{circumflex over ( )}10 cells Gelatin: ETEC Cells 132.52 mg: 1
.times. 10{circumflex over ( )}10 cells D-sorbitol: ETEC Cells
13.23 mg: 1 .times. 10{circumflex over ( )}10 cells Alpha-Gal-Cer:
ETEC Cells 0.41 mg: 1 .times. 10{circumflex over ( )}10 cells Note:
the above calculated figures are an approximation which disregards
the mass of water in the ETEC suspension and the mass of any
residual water in the beads.
[0421] The teaching of this paragraph applies to the entire
disclosure of the application, including all compatible embodiments
of the description and all compatible claims. The invention
includes compositions having a Feature selected from Feature 1,
Feature 2, Feature 3 and Feature 4 of the following table. In the
table, the word "cells" refers to the microorganism cells of the
composition concerned, e.g. cells of any bacterium, fungus or
unicellular pathogen referred to herein.
TABLE-US-00006 Ratio Feature (dry wt mg to 10{circumflex over (
)}10 cells) Range 1 Range 2 1 Surfactant: Cells 25-125 mg:
10{circumflex over ( )}10 cells 50-100 mg: 10{circumflex over (
)}10 cells 2 Hydrogel-forming polymer: 75-175 mg: 10{circumflex
over ( )}10 cells 100-150 mg: 10{circumflex over ( )}10 cells Cells
3 Plasticiser: Cells 2-25 mg: 10{circumflex over ( )}10 cells 5-20
mg: 10{circumflex over ( )}10 cells 4 Adjuvant: Cells 0.1-10 mg:
10{circumflex over ( )}10 cells 0.25-5 mg: 10{circumflex over (
)}10 cells
[0422] The invention across its entire disclosure (as described in
the preceding paragraph) further includes compositions having any
one of the following combinations of Features of the above table:
[0423] For Range 1: Feature 1 & Feature 2; Feature 1 &
Feature 3; Feature 1 & Feature 4; Feature 2 & Feature 3;
Feature 2 & Feature 4; Feature 3 & Feature 4; Feature 1
& Feature 2 & Feature 3; Feature 1 & Feature 3 &
Feature 4; Feature 1 & Feature 2 & Feature 4; Feature 2
& Feature 3 & Feature 4; Feature 1 & Feature 2 &
Feature 3 & Feature 4. For Range 2: Feature 1 & Feature 2;
Feature 1 & Feature 3; Feature 1 & Feature 4; Feature 2
& Feature 3; Feature 2 & Feature 4; Feature 3 & Feature
4; Feature 1 & Feature 2 & Feature 3; Feature 1 &
Feature 3 & Feature 4; Feature 1 & Feature 2 & Feature
4; Feature 2 & Feature 3 & Feature 4; Feature 1 &
Feature 2 & Feature 3 & Feature 4.
[0424] The invention across its entire disclosure further includes
compositions having any of the following ratios of adjuvant to
cells (dry wt mg to 10 10 cells): 0.1-100 mg:10 10 cells, 0.2-10
mg:10 10 cells, 0.25-10 mg:10 10 cells; 0.4-10 mg:10 10 cells;
0.2-5 mg:10 10 cells, 0.25-5 mg:10 10 cells; 0.4-5 mg:10 10 cells;
1-10 mg:10 10 cells, 2-10 mg:10 10 cells; 4-10 mg:10 10 cells;
10-100 mg:10 10 cells, 25-100 mg:10 10 cells; 50 mg-100 mg:10 10
cells; 10-50 mg:10 10 cells, 10-30 mg:10 10 cells; 10 mg-20 mg:10
10 cells. The teaching of this paragraph applies e.g. to each of
Features 1, 2 and 3 in the above table in respect of Ranges 1 and 2
and to combinations of two or three of Features 1, 2 and 3
mentioned in the immediately preceding paragraph. The microorganism
may consist of one or more unicellular microorganisms, for example
selected from bacteria and unicellular fungi, and the ratio of
adjuvant to the aggregate amount of unicellular microorganisms (mg
dry weight of adjuvant to 10 10 cells) may in this case be as
recited earlier in this paragraph and elsewhere herein. The
microorganism may comprise or be any one or more, e.g. one, two or
three, microorganisms mentioned in this specification. The
microorganism may comprise or be any one or more, e.g. one, two or
three, bacteria mentioned in this specification. The microorganism
may comprise or be ETEC. The microorganism may comprise or be h.
pylori. The microorganism may comprise or be a V. Cholerae.
[0425] The invention across its entire disclosure therefore
includes compositions in which the microorganism consists of one or
more unicellular microorganisms. In such compositions, the ratio of
surfactant to the aggregate amount of unicellular microorganisms
(mg dry weight of surfactant to 10'10 cells) may be, for example,
from 10-200 mg:10 10 cells and optionally from 25-125 mg:10 10
cells, e.g. from 25-150 mg:10 10 cells, 25-100 mg:10 10 cells,
50-200 mg:10 10 cells, 50-100 mg:10 10 cells or 60-90 mg:10 10
cells.
[0426] The dry weight ratio of adjuvant to surfactant (Kolliphor
HS) in the examples is about 175 to 1, and this is an optional
ratio applicable across the entire scope of the disclosure. Also to
be mentioned as applicable across the entire scope of the
disclosure are optional ratios of 200 to 1 or less, 150 to 1 or
less, 100 to 1 or less and 50 to 1 or less.
[0427] Beads were coated on MFL01 to achieve 5.5% weight gain of
Eudragit L 30 D-55.
[0428] A summary of Formulations 1 and 2 prepared is presented in
the table below.
TABLE-US-00007 Kolliphor HS15 Weight to Gelatin gain of w/w ratio L
30 D 55 Formulation Antigen Adjuvant(s) (dry weight) achieved 1
ETEC (2.76 .times. None 1:1.60 5.9% (comparative) 10{circumflex
over ( )}8 cells per dose (3 beads)) 2 ETEC (2.78 .times. aGalCer
1:1.66 5.5% 10{circumflex over ( )}8 cells per (0.18%) dose (3
beads))
[0429] The antigen shown in the above table (ETEC) comprised
formalin-killed E. coli K12 bacteria (expressing CFA/I fimbriae on
the surface).
[0430] The above compositions were used to determine whether and to
what extent oral immunisation of mice with these compositions (with
and without alpha-GalCer as adjuvant) would induce potent mucosal
and systemic antibody responses.
[0431] Female BALB/c mice were immunised by oral gavage on days 0
and 1 with bicarbonate buffer as a control or with ETEC
(3.times.10.sup.8 cells/dose) in bicarbonate buffer either alone or
together with alpha-GalCer (10 .mu.g/dose) or cholera toxin (CT; 10
.mu.g/dose) as adjuvant or immunized orally with SmPill.RTM.
containing ETEC (3.times.10.sup.8 cells/dose) alone or together
with alpha-GalCer (10 .mu.g/dose). Mice were boosted on days 13,
14, 27 and 28 with an identical series of immunizations. Groups of
mice (n=5) were bled 1 day prior to booster immunizations and 12
days after the final dose for determination of IgG and IgA antibody
titres. Faecal pellets were collected from all mice 1 day prior to
booster immunizations and 12 days after the final immunization to
determine mucosal antibody responses. Two weeks post the final
immunization, mice were sacrificed and intestinal washes were
collected. Extracts of both the small and large intestines of mice
were also obtained and frozen in buffer with protease inhibitors
for subsequent analysis of mucosal antibodies using saponin to
extract proteins from the intestines.
[0432] Experimental Groups
1. Bicarbonate Buffer
2. ETEC
[0433] 3 ETEC+alpha-GalCer
4. ETEC+CT
5. SmPill.RTM. (ETEC)
[0434] 6. SmPill.RTM. (ETEC+alpha-GalCer)
[0435] Enhanced antibody responses were found in the sera, faecal
pellets, saliva and intestinal washes of mice immunized with
SmPill.RTM. containing ETEC and alpha-GalCer compared to mice
immunized with non-adjuvanted SmPill.RTM. vaccine or with ETEC in
solution.
Results on ETEC Compositions
Please Refer to FIGS. 1 to 8
[0436] ETEC alone in solution: Oral immunisation of mice with the
ETEC vaccine in solution did not induce an antigen-specific IgA
antibody response; CFA/I-specific IgA was only detectable in the
faecal pellets (FIG. 2) or serum (FIG. 4) of 1/5 mice immunized
with this vaccine.
[0437] ETEC+alpha-GalCer in solution: The addition of alpha-GalCer
as adjuvant in solution did not result in significantly enhanced
antigen-specific antibody titres either mucosally or systemically
compared to ETEC alone in solution.
[0438] SmPill.RTM. (ETEC alone): The delivery of the ETEC vaccine
in a non-adjuvanted SmPill.RTM. formulation did not result in
detectable mucosal antibodies in the faecal pellets (FIGS. 1 and 2)
or the intestines (FIGS. 6, 7 and 8) of any immunized mice.
Furthermore, systemic antibody responses were not significantly
enhanced in mice immunized with this formulation compared to those
administered the ETEC vaccine in solution orally (FIGS. 3 and 4).
SmPill.RTM. (ETEC+alpha-GalCer): The delivery of ETEC in
SmPill.RTM. together with alpha-GalCer as adjuvant induced a
specific IgA response in the faecal pellets (FIG. 2), the sera
(FIG. 4) and the intestines (FIG. 7) of all immunized mice after
the final oral immunization. Faecal pellets from mice immunized
with this SmPill.RTM. formulation contained significantly higher
mucosal IgA and IgG (FIG. 1) compared to either ETEC+alpha-GalCer
in solution or ETEC alone in SmPill.RTM.. While specific IgA
antibodies were detected after the second series of vaccinations,
the enhancing effect of the SmPill.RTM. formulation was most
clearly seen after the 3.sup.rd series of immunizations.
Furthermore, following extraction of intestinal proteins by
saponin, significantly greater antigen-specific IgG and IgA
antibody titres were found in the intestines of mice immunized
orally with SmPill.RTM. containing ETEC and alpha-GalCer compared
to mice administered non-adjuvanted vaccine either in solution or
in SmPill.RTM. (FIGS. 7 and 8). Significantly enhanced
CFA/I-specific IgG antibodies were also found in the small
intestinal washes of mice immunized with SmPill.RTM. containing
ETEC and alpha-GalCer (FIG. 6).
[0439] In terms of systemic IgA and IgG, the strongest responses
were again detected in mice immunized orally with SmPill.RTM.
containing ETEC and alpha-GalCer (FIGS. 3, 4 and 5).
Antigen-specific IgG antibodies were significantly greater from the
sera of mice immunized with ETEC and alpha-GalCer in SmPill.RTM.,
following either two or three series of oral immunizations,
compared to either ETEC+alpha-GalCer in solution or ETEC alone in
SmPill.RTM. (FIG. 3). Analysis of the antibody subclasses induced
by this formulation revealed that IgG1 was the main subtype induced
(FIG. 5).
[0440] Conclusions [0441] Oral immunization of mice with ETEC
formulated in SmPill.RTM. containing alpha-GalCer induced
significantly stronger mucosal IgA and IgG antibody responses in
the faecal pellets and intestines compared to immunization with
ETEC in solution or ETEC in SmPill.RTM. without alpha-GalCer.
[0442] Immunization of mice with SmPill.RTM. containing ETEC and
alpha-GalCer also induced antigen-specific antibody responses in
the serum, which were significantly greater than those generated in
mice immunized orally with non-adjuvanted ETEC vaccine either in
solution or in SmPill.RTM.. In summary, these data confirm that a
SmPill.RTM. formulation containing ETEC and alpha-GalCer as
adjuvant according to the invention induces potent mucosal and
systemic antibody responses. Furthermore, this study shows that by
extraction of intestinal proteins using saponin, antigen-specific
antibodies could be detected from the intestines of mice immunized
orally with SmPill.RTM. containing ETEC and alpha-GalCer according
to the invention and that these responses were significantly
greater than those induced by non-adjuvanted vaccine.
[0443] V Cholerae Compositions
[0444] In the following examples the microorganism is a formalin
killed genetically engineered a strain of JS1569 V. Cholerae which
over expresses Inaba LPS.
[0445] References to "JS1569" and "JS1569 V. Cholerae" in the
examples and figures refer to the above mentioned engineered strain
of JS1569 V. Cholerae which over expresses Inaba LPS.
[0446] JS1569 V. Cholerae SmPill.RTM. Formulations
[0447] Formulations 3 and 4 were prepared using analogous methods
to those described above in relation to the ETEC formulations to
give the following compositions
TABLE-US-00008 % Weight Gain of Eudragit V.Cholerae Adjuvant dose L
30 D dose (2 beads) Adjuvant (2 beads) 55 Formulation 3 2.6 .times.
10.sup.8 None N/A 5.4 (comparative) Formulation 4 2.5 .times.
10.sup.8 .alpha.-GalCer 10 .mu.g 7.4
[0448] Briefly, Formulations 3 and 4 were prepared as follows:
[0449] Formulation 3 (V. Cholerae)
[0450] 1.40 g of V. Cholerae aqueous suspension
(conc.=2.24.times.10.sup.10 bacteria per ml) were mixed with 0.41 g
of Kolliphor HS 15 at room temperature until a homogeneous
dispersion was obtained ("Surfactant phase"). Surfactant phase
contained 22.1% of Kolliphor HS 15 and 77.9% of V. Cholerae aqueous
suspension (% by weight).
[0451] In a different vial, 0.71 g of V. Cholerae aqueous
suspension (conc.=2.24.times.10.sup.10 bacteria per ml) were mixed
with 2.3 g of purified water; 0.063 g of Sorbitol were added and
the solution was mixed until all Sorbitol dissolved. 0.63 g of
gelatin was added and temperature was increased up to 55.degree.
C.; the mixture was stirred until all gelatin melted ("Gelatin
phase"). Gelatin phase consisted of 17.1% gelatin, 1.7% sorbitol,
62.1% purified water and 19.1% V. Cholerae suspension (% by
weight).
[0452] The surfactant phase was added into the gelatin phase; the
resulting composition was mixed at 55.degree. C. until a
homogeneous combined phase was obtained; the composition was then
ejected through a 1 mm nozzle into medium chain triglyceride oil
cooled at 4.degree. C. to form spherical beads. After all beads
were produced, the cooling oil was drained out and the beads were
dried at room temperature overnight.
[0453] The expected dry composition of the emulsion batch prior to
bead formation was as follows:
TABLE-US-00009 weights (g) % w/w V.Cholerae cells 4.7 .times.
10.sup.10 cells Kolliphor HS 15 0.4104 37.1 Gelatin 0.6325 57.2
Sorbitol 0.0634 5.7 Total 1.1063
[0454] The composition in the above table represents that of the
total batch prior to bead formation. Accordingly the average number
of cells per bead may be determined based upon the e average bead
size and weight as described above in relation to Formulations 1
and 2 above.
[0455] The beads were then coated with 5.4% (weight gain) Eudragit
L-30 D 55. The resulting composition contained V. Cholerae bacteria
as antigen at a dose of 1.3.times.10.sup.8 bacteria per bead.
[0456] Formulation 4 (V. Cholerae and .alpha.-GalCer)
[0457] 1.41 g of V. Cholerae aqueous suspension
(conc.=2.24.times.10.sup.10 bacteria per ml) were mixed with 0.41 g
of Kolliphor HS 15 and 2 mg of .alpha.-GalCer at room temperature
until a homogeneous dispersion was obtained ("Surfactant phase").
Surfactant phase contained 22.1% of Kolliphor HS 15, 0.1% of
.alpha.-GalCer and 77.8% of V. Cholerae aqueous suspension (% by
weight).
[0458] In a different vial, 0.72 g of V. Cholerae aqueous
suspension (conc.=2.24.times.10.sup.10 bacteria per ml) were mixed
with 2.3 g of purified water; 0.064 g of Sorbitol were added and
the solution was mixed until all Sorbitol dissolved. 0.63 g of
gelatin was added and temperature was increased up to 55.degree.
C.; the mixture was stirred until all gelatin melted ("Gelatin
phase"). Gelatin phase consisted of 17.1% gelatin, 1.7% sorbitol,
61.8% purified water and 19.4% V. Cholerae suspension (% by
weight).
[0459] Surfactant phase was added into the gelatin phase; the
resulting composition was mixed at 55.degree. C. until a
homogeneous phase was obtained. The composition was then formed
into beads and dried as described in Formulation 3.
[0460] The expected dry composition of the emulsion batch prior to
bead formation was as follows:
TABLE-US-00010 weights (g) % w/w V.Cholerae cells 4.7 .times.
10.sup.10 cells .alpha.GalCer 0.0020 0.2 Kolliphor HS 15 0.3999
36.4 Gelatin 0.6335 57.6 Sorbitol 0.0640 5.8 Total 1.0994
[0461] The beads were coated with 7.4% (weight gain) Eudragit L-30
D 55. The beads contained V. Cholerae bacteria as antigen at a
theoretical dose of 1.25.times.10.sup.8 bacteria per bead plus
.alpha.-GalCer as adjuvant at a theoretical dose of 5.3 .mu.g per
bead.
[0462] Formulations 5, 6 and 7 and Comparative Formulations A and
B
[0463] The following compositions were prepared using an analogous
method to that used for the preparation of Formulations 3 and
4.
TABLE-US-00011 % Weight Gain of Eudragit V.Cholerae dose Adjuvant
dose L 30 D Formulation (3 beads) Adjuvant (3 beads) 55 Comparative
A 4.5 .times. 10.sup.8 None -- 7.4 Formulation 5 4.5 .times.
10.sup.8 .alpha.-GalCer 10 .mu.g 6.1 Formulation 6 4.5 .times.
10.sup.8 Cholera Toxin 10 .mu.g 6.9 Formulation 7 12 .times.
10.sup.8 .alpha.-GalCer 10 .mu.g 8.1 Comparative B 12 .times.
10.sup.8 None -- 9.1
[0464] Formulations 3 and 4 were used in Experiments 1 to 3 below.
Formulations 5, 6, 7 and Comparative formulations A and B were used
in Experiments 4 and 5.
[0465] Mice and Dosing
[0466] Female C57/bl6 Mice (Harlan UK) were used in the studies.
The mice were immunised with solutions and/or the SMPill.RTM.
compositions as follows. Solutions: Anesthetised mice were gavaged
with 200 .mu.l of solution containing the bacteria+/-adjuvant in
bicarbonate solution (0.35M at pH 9). SmPill.RTM. compositions:
SmPill.RTM. compositions (generally two beads) were administered to
anesthetised mice by flexible gavage needles in a small volume of
delivery buffer (PBS at pH 5).
[0467] V. Cholerae Experiment 1: Immune Response in Fecal
Pellets
Mice were orally vaccinated with sodium bicarbonate solution alone
or containing 2.5.times.10.sup.8 bacteria with/without 10 .mu.g
cholera toxin or 10 .mu.g .alpha.GalCer; or 2.5.times.10.sup.8
bacteria with/without 10 .mu.g .alpha.GalCer in SmPill.RTM.
formulations on two consecutive days at 2 week intervals for a
total of 3 rounds. Fecal pellet samples were obtained the day
before each round of vaccination and 2 weeks after the final
vaccination and anti Inaba-LPS IgA and IgG responses measured.
Fecal pellet samples were obtained the day before each round of
vaccination and 2 weeks after the final vaccination and anti
Inaba-LPS IgA and IgG responses measured. FIG. 13 shows the IgA
titres observed in fecal pellets (a) after 1 round, (b) after 2
rounds, (c) after 3 rounds of immunisation and (e) shows the IgG
titre in fecal pellets after 3 rounds of immunisation which were
measured by ELISA. Results in FIGS. 13 (a) to (c) and (e) represent
the mean and SD of all mice in each group. FIG. 14 shows that
corresponding time course and illustrates the kinetics of the
induction of the IgA response after each round of vaccination.
V. Cholerae Experiment 2: Immune Response Locally in the
Intestine
[0468] Mice were orally vaccinated as described in V. Cholerae
Experiment 1. 2 weeks after the final round of vaccination mice
were euthanized and perfused to remove the blood from the internal
organs. The small intestine was dissected out and a small portion
of the upper section removed that placed into a saponin solution to
extract the inter tissue antibodies. FIG. 15 shows the Inaba
specific LPS IgA (a) and IgG (b) titres measured by ELISA. Results
represent the mean and SD of all mice in each group.
[0469] V. Cholerae Experiment 3: Systemic Immune Response
[0470] Mice were orally vaccinated as described in V. Cholerae
Experiment 1. 2 weeks after the final round of vaccination serum
was obtained by tail bleeds. FIG. 16 shows Inaba specific LPS IgA
(a), IgG (b) and IgG1 (c) titres measured by ELISA. Results
represent the mean and SD of all mice in each group.
[0471] V. Cholerae Experiment 4: High Dose Immunisation--Fecal
Pellets
[0472] Mice were orally vaccinated with placebo (gelatin)
SmPill.RTM. or SmPills containing i) 4.5.times.10.sup.8 bacteria
without adjuvant; ii) 4.5.times.10.sup.8 bacteria and 10 .mu.g
cholera toxin; iii) 4.5.times.10.sup.8 bacteria with 10 .mu.g
.alpha.GalCer; or SmPills with 12.times.10.sup.8 bacteria
with/without 10 .mu.g .alpha.GalCer for either one or two
consecutive days at 2 week intervals for a total of 3 rounds. Fecal
pellet samples were obtained the day before each round of
vaccination and 2 weeks after the final vaccination and anti
Inaba-LPS IgA and IgG responses measured. FIG. 17 shows the various
IgA titres observed after 1, 2 and 3 rounds of immunisation
measured by ELISA. Results represent OD492 absorbance values over a
series of dilutions of the sample.
[0473] V. Cholerae Experiment 5: High Dose Immunisation--Intestinal
IgA Response
[0474] Mice were orally vaccinated as described in V. Cholerae
Experiment 4. 2 weeks after the final round of vaccination mice
were euthanized and perfused to remove the blood from the internal
organs. The small intestine was dissected out and a small portion
of the upper section removed that placed into a saponin solution to
extract the inter tissue antibodies. FIG. 18 show the various Inaba
LPS-specific IgA titres observed after 3 rounds of immunisation
which were measured by ELISA. Results represent OD492 absorbance
values over a series of dilutions of the sample.
[0475] Conclusions--Please Refer to FIGS. 13 to 18
[0476] SmPill.RTM. compositions containing .alpha.GalCer and JS1569
V. Cholerae show an enhanced immune response compared to
compostions containing JS1569 V. Cholerae alone illustrating that
.alpha.GalCer is an effective adjuvant for SmPill compositions
containing a microorganism (FIGS. 13 to 18).
[0477] SmPill.RTM. compositions containing JS1569 together with
.alpha.GalCer induce stronger responses systemically and locally in
the intestine than the same formulation delivered in solution or
solutions of the antigen alone or the antigen and CT (FIGS. 15 and
16).
[0478] SmPill.RTM. compositions containing JS1569 together with
.alpha.GalCer induce stronger antibody titres in the fecal material
than the same formulation delivering the antigen alone (FIG.
17).
[0479] Vaccination on two consecutive days with SmPill.RTM.
compositions containing .alpha.GalCer and JS1569 V. Cholerae shows
no benefit over vaccination just one day per round of vaccination
(FIGS. 17 and 18).
[0480] Reference Studies 1 and 2
[0481] SmPill.RTM. compositions comprising Cholera toxin subunit B
(CTB) as antigen with alpha-GalCer were used in Reference Studies 1
and 2 in mice, the results of which are shown in FIGS. 10 to 12.
The compositions were prepared using analogous methods to those
described above to give the following expected dry weight
composition.
TABLE-US-00012 weights (g) % w/w CTB 0.0075 0.67 Kolliphor HS 15
0.4129 36.79 Gelatin 0.6365 56.72 .alpha.-GalCer 0.0020 0.18
Sorbitol 0.0633 5.64 Total 1.1222
Reference Studies 1 and 2 show the effect of a composition
containing a sub-unit antigen as opposed to the "whole-cell"
microorganism according to the present invention.
Aspect Ratio
[0482] Minibeads were made generally following the above-described
procedures by extrusion from a nozzle to fall into a cooling
medium. In this instance, though, the beads did not fall within the
invention in having a microorganism as active Some of the beads
were then coated as described herein with a Surelease.TM. and
pectin mixture. Sample populations of the coated beads and sample
populations of uncoated beads were both typically found to have an
average aspect ratio of 1.2 when measured using an Eyecon.TM.
particle characteriser.
* * * * *