U.S. patent application number 10/620686 was filed with the patent office on 2005-07-28 for biodegradable targetable microparticle delivery system.
Invention is credited to Chong, Pele, Klein, Michel H., Sokoll, Kenneth K..
Application Number | 20050163745 10/620686 |
Document ID | / |
Family ID | 25089888 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050163745 |
Kind Code |
A1 |
Sokoll, Kenneth K. ; et
al. |
July 28, 2005 |
Biodegradable targetable microparticle delivery system
Abstract
Copolymers designed for use as particulate carriers containing
functionalizable amino acid subunits for coupling with targeting
ligands are described. The copolymers are polyesters composed of
.alpha.-hydroxy acid subunits such as D,L-lactide and
pseudo-.alpha.-amino acid subunits which may be derived from serine
or terpolymers of D,L-lactide and glycolide and
pseudo-.alpha.-amino acid subunits which may be derived from
serine. Stable vaccine preparations useful as delayed release
formulations containing antigen or antigens and adjuvants
encapsulated within or physically mixed with polymeric
microparticles are described. The particulate carriers are useful
for delivering agents to the immune system of a subject by mucosal
or parenteral routes to produce immune responses, including
antibody and protective responses.
Inventors: |
Sokoll, Kenneth K.; (Alton,
CA) ; Chong, Pele; (Richmond Hill, CA) ;
Klein, Michel H.; (Toronto, CA) |
Correspondence
Address: |
Michael I. Stewart
Sim & McBurney, 6th Floor
330 University Avneue
Toronto
ON
M5G 1R7
CA
|
Family ID: |
25089888 |
Appl. No.: |
10/620686 |
Filed: |
July 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10620686 |
Jul 17, 2003 |
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09331118 |
Aug 31, 1999 |
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6623764 |
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09331118 |
Aug 31, 1999 |
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PCT/CA97/00980 |
Dec 19, 1997 |
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09331118 |
Aug 31, 1999 |
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08770850 |
Dec 20, 1996 |
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6042820 |
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Current U.S.
Class: |
424/78.37 ;
525/54.1 |
Current CPC
Class: |
Y10S 530/815 20130101;
Y10S 514/952 20130101; A61K 9/1647 20130101; A61K 9/167 20130101;
A61K 2039/55555 20130101; C08G 63/6852 20130101; Y10S 530/816
20130101; Y10T 428/2982 20150115 |
Class at
Publication: |
424/078.37 ;
525/054.1 |
International
Class: |
A61K 031/785 |
Claims
1-70. (canceled)
71. An immunogenic composition comprising a particulate carrier for
delivery of biologically active material to a host, an immunogen
and a physiologically acceptable carrier thereof, said carrier
comprising a polymer having a molecular weight of about 5000 to
about 40,000 daltons and having the general formula: wherein:
R.sub.1, R.sub.2 and R.sub.4 are selected independently and are
selected from H.sub.1, linear or 3branched alkyl groups; R.sub.3
and R.sub.4 are H; R.sub.6 is selected from H, an amine protecting
group, a spacer molecule or a biologically active species; X is
selected from an O or S group; and x and y are integers.
72. A method of producing an immune response in a host comprising
administering the immunogenic composition of claim 71 to said
host.
73. The method of claim 72, wherein said composition is
administered mucosally or parenterally.
74. The method of claim 72, wherein said immune response is an
antibody response.
75. The method of claim 74, wherein said antibody response is a
local or serum antibody response.
76. The immunogenic composition of claim 71 wherein said
particulate carrier has a particle size of about 1 to 10 .mu.m.
77. The composition of claim 71, wherein said polymer is derived by
copolymerization of monomers comprising at least one
.alpha.-hydroxy acid and at least one pseudo-.alpha.-amino
acid.
78. The composition of claim 77, wherein the at least one
.alpha.-hydroxy acid has the formula of R.sub.1R.sub.2COHCO.sub.2H,
wherein the R.sub.1 and R.sub.2 groups are H, linear or branched
alkyl units, the alkyl unit being represented by the formula
C.sub.nH.sub.2n+1, where n=integer of about 1 to 10.
79. The composition of clam 78, wherein said .alpha.-hydroxy acids
comprise a mixture of .alpha.-hyrdroxy acids, one of said mixture
of .alpha.-hyrdoxy acids having R.sub.1 and R.sub.2 groups which
are hydrogen and the other of said mixture of .alpha.-hydroxy acids
having an R.sub.1 group which is CH.sub.3 and R.sub.2 group which
is H.
80. The composition of claim 77, wherein the at least one
pseudo-.alpha.-hydroxy acids has the formula
R.sub.5CHNHR.sub.6CO.sub.2H, wherein the R.sub.5 group is a
hydroxylmethyl or methyl thiol group and R.sub.6 is an amine
protecting group.
81. The composition of claim 80, wherein the amine protecting group
is selected from the group consisting of carbobenzyloxy (CBZ or Z)m
benzyl (Bn)m paramethoxybenzyl (MeOBn), benzyloxymethoxy (BOM),
tert-butyloxycarbonyl (t-BOC) and [9-fluorenylmethyl oxy]carbonyl
(FMOC).
82. The composition of claim 77, wherein the at least one
.alpha.-hydroxy acid is selected from the group consisting of
L-lactic acid, D,L-lactic acid, glycolic acid, hydroxy valeric acid
and hydroxybutyric acid.
83. The composition of claim 77, wherein the at least one
pseudo-.alpha.-amino acid is derived from serine.
84. The composition of claim 71, wherein said at least one
.alpha.-hydroxy acid monomer and at least one pseudo-.alpha.-amino
acid monomer are selected to result in poly-D,
L-lactide-co-glycolide-co-pseudo-Z-serine ester (PLGpZS).
85. The composition of claim 71, wherein said at least one
.alpha.-hydroxy acid monomer and at least one pseudo-.alpha.-amino
acid monomer are selected to result in poly-D,
L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS).
86. The composition of claim 71, wherein R.sub.6 is at least one
biologically active species.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of copending U.S.
patent Ser. No. 08/770,050 filed Dec. 20, 1996.
FIELD OF THE INVENTION
[0002] The present invention relates to biodegradable
microparticles for delivery of a biologically active material and
is particularly concerned with such microparticles that are
targetable to particular cell types.
BACKGROUND OF THE INVENTION
[0003] Vaccines have been used for many years to protect humans and
animals against a wide variety of infectious diseases. Such
conventional vaccines consist of attenuated pathogens (for example,
polio virus), killed pathogens (for example, Bordetella pertussis)
or immunogenic components of the pathogen (for example, diphtheria
toxoid and hepatitis B surface antigen).
[0004] Some antigens are highly immunogenic and are capable alone
of eliciting protective immune responses. Other antigens, however,
fail to induce a protective immune response or induce only a weak
immune response. The immune response of a weakly immunogenic
antigen can be significantly enhanced if the antigens are
co-administered with adjuvants. Adjuvants enhance the
immunogenicity of an antigen but are not necessarily immunogenic
themselves. Adjuvants may act by retaining the antigen locally near
the site of administration to produce a depot effect facilitating a
slow, sustained release of antigen to cells of the immune system.
Adjuvants can also attract cells of the immune system to an antigen
depot and stimulate such cells to elicit immune responses.
Adjuvants have been identified that enhance the immune response to
antigens delivered parenterally.
[0005] Adjuvants are commonly employed with antigen in vaccine
formulations whereby the induction of systemic immunity through
parenteral immunization (intramuscular or subcutaneous) is
obtained. This approach is suitable for infectious agents gaining
access to the body via damaged skin (i.e. Tetanus), however, there
are problems encountered due to side-effects and associated
toxicity of many adjuvants administered in this fashion. Only those
vaccines formulated from aluminum salts (aluminum phosphate or
aluminum hydroxide) find routine use in human and veterinary
vaccination. However, even these adjuvants are not suitable for use
with all antigens and can also cause irritation at the site of
injection. There is a clear need to develop adjuvants which safely
enhance the immunogenicity of antigens at the site of
injection.
[0006] There are other problems specific to the nature of the
antigen being used. For example, most conventional non-living
vaccines require multiple doses for effective immunization. Live
attenuated vaccines and many nonliving liquid vaccines suffer from
the need for controlled storage conditions and are susceptible to
inactivation (e.g. thermal sensitivity). There are also problems
associated with combining vaccines in single dosage forms, due to
adjuvant incompatibilities, pH, buffer type and the presence of
salts.
[0007] Mucosal immunity is induced primarily by induction of
secretory immunoglobulin (sIgA) in intestinal, bronchial or nasal
washings and other external secretions. For example, parenteral
cholera vaccines have been shown to offer limited protection
whereas the more recently developed oral form is highly effective
(ref. 1--throughout this specification, various references are
referred to in parenthesis to more fully describe the state of the
art to which this invention pertains. Full bibliographic
information for each citation is found at the end of the
specification, immediately preceding the claims. The disclosures of
these references are hereby incorporated by reference into the
present disclosure). Studies with human volunteers have shown that
oral administration of influenza vaccine is effective at inducing
secretory anti-influenza antibodies in nasal secretions and
substances have been identified which might be useful as adjuvants
for such ingested vaccines. However, most of these adjuvants are
relatively poor in terms of improving immune responses to ingested
antigens. Currently, most of these adjuvants have been determined
to be safe and efficacious in enhancing immune responses in humans
and animals to antigens that are administered via the
orogastrointestinal, nasopharyngeal-respiratory and genital tracts
or in the ocular orbits. However, administration of antigens via
these routes is generally ineffective in eliciting an immune
response. Although the above example illustrates the potential of
these immunization modes, the development of vaccine formulations
for use by these routes has been slow for various reasons. The
inability to immunize at the mucosal surface is generally believed
to be due to include:
[0008] (i) antigen degradation via the acid and/or proteolytic
enzymes present during the transit to the mucosal surfaces;
[0009] (ii) antigen degradation by secretions presented at the
mucosal epithelium;
[0010] (iii) limited adsorption across the mucosal epithelium;
[0011] (iv) the dilution of the antigen to a concentration that is
below that required to induce immune responses; and
[0012] (v) ineffective adjuvants and/or delivery systems.
[0013] The problems associated with the use of adjuvants in
parenteral vaccine formulations and the lack of suitable systems
for vaccine delivery to mucosal sites understates the need for new
techniques that are effective when administered by various routes
and are inherently free from associated toxicity concerns or
side-effects.
[0014] It is also desired to provide vaccine delivery in a single
dosage form for both human and animal immunizations as this has the
advantage of reducing time and cost, and in human medicine,
increases patient compliance which is of extreme importance in
developing countries where access is restricted. This is especially
true for infants within these countries.
[0015] In order to increase immune responses to administered
antigens, a carrier may be used to protect the antigen from
degradation and also modulate the uptake of these materials in
vivo. Sensitive antigens may be entrapped to protect them against
destruction, reduction in immunogenicity or dilution. Methods for
formulating a carrier include dispersing an antigen within a
polymeric matrix (monolithic matrix) or by the coating of a
polymeric material around an antigen to give an outer protective
wall (core-shell). The manipulation of the formulation protocol can
allow for control over the average size of these materials. This
has been shown to be important for the uptake of particulates via
oral delivery at specialized M-cells of the Peyers patches within
the intestinal tract.
[0016] U.S. Pat. No. 5,151,264 describes a particulate carrier of a
phospholipid/glycolipid/polysaccharide nature that has been termed
Bio Vecteurs Supra Moleculairs (BVSM). The particulate carriers are
intended to transport a variety of molecules having biological
activity in one of the layers thereof. However, U.S. Pat. No.
5,151,264 does not describe particulate carriers containing
antigens for immunization and particularly does not describe
particulate carriers for immunization via the orogastrointestinal,
nasapharyngeal-respiratory and urogenital tracts and in the ocular
orbits or other mucosal sites.
[0017] Eldridge et al. (refs 2 and 3) observed the delayed release
of antigen in vivo from biodegradable microspheres manufactured
from polylactide-co-glycolide copolymer also known as PLG or PLGA.
Numerous other polymers have been used to encapsulate antigens for
formulation into microparticles and some of these include
polyglycolide, polylactide, polycaprolactone, polyanhydrides,
polyorthoesters and poly(.alpha.-hydroxybutyric acid).
[0018] U.S. Pat. No. 5,075,109 describes encapsulation of the
antigens trinitrophenylated keyhole limpet hemocyanin and
staphylococcal enterotoxin B in 50:50
poly(DL-lactide-co-glycolide). Other polymers for encapsulation are
suggested, such as poly(glycolide), poly(DL-lactide-co-glycolide),
copolyoxalates, polycaprolactone, poly(lactide-co-caprolactone),
poly(esteramides), polyorthoesters and poly(.alpha.-hydroxybutyric
acid), and poly anhydrides. The encapsulated antigen was
administered to mice via gastric intubation and resulted in the
appearance of significant antigen-specific IgA antibodies in saliva
and gut secretions and in sera. As is stated in this patent, in
contrast, the oral administration of the same amount of
unencapsulated antigen was ineffective at inducing specific
antibodies of any isotype in any of the fluids tested.
Poly(DL-lactide-co-glycolide) microcapsules were also used to
administer antigen by parenteral injection.
[0019] Published PCT application WO 91/06282 describes a delivery
vehicle comprising a plurality of bioadhesive microspheres and
antigenic vaccine ingredients. The microspheres being of starch,
gelatin, dextran, collagen or albumin. This delivery vehicle is
particularly intended for the uptake of vaccine across the nasal
mucosa. The delivery vehicle may additionally contain an absorption
enhancer. The antigens are typically encapsulated within protective
polymeric materials.
[0020] U.S. Pat. No. 5,571,531 describes particulate carriers
comprising a solid matrix of a polysaccharide and a proteinaceous
material. A functionalized silicone polymer is bonded to the matrix
for the delivery of materials having biological activity.
[0021] Although time-delayed release of antigen was shown in the
above work, difficulties were encountered when microparticles are
manufactured by the described methods. The exposure of biological
materials to the organic solvents and physical forces used can lead
to denaturation. It may be also be difficult to scale-up the
procedures. Furthermore, hydrophilic antigens may be inefficiently
encapsulated.
[0022] It would be desirable to provide improved carriers without
such limitations. It would be particularly desirable to provide
polymeric materials which can be formulated into microparticles and
microspheres and which contain targeting moieties to target the
antigen to preselected ligands. This would have tremendous
potential for cells of the immune system.
SUMMARY OF THE INVENTION
[0023] The present invention is directed towards the production of
a novel and useful polymer that has properties suitable for
manufacturing by various processes into microparticles and
microspheres. In this invention, modifications of existing
processing procedures results in significant improvement in
encapsulation efficiencies.
[0024] This invention is further directed to the production of
useful vaccine delivery systems for antigen(s) or antigen and
co-adjuvant cocktails by various immunization routes which include
parenteral, oral and intranasal.
[0025] In accordance with a first aspect of the invention, there is
provided a novel biodegradable, biocompatible polymer, including
those having a molecular weight of about 5,000 to about 40,000
daltons, having a backbone of the general formula: 1
[0026] wherein;
[0027] R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are selected
independently and are selected from H, linear or branched alkyl
groups;
[0028] R.sub.6 is selected from H, an amine protecting group, a
spacer molecule or a biologically active species;
[0029] X is selected from an O or S group; and
[0030] x and y are integers, including values such that at least
about 95% of the polymer is comprised of .alpha.-hydroxy acid
residues.
[0031] The novel polymers are derived by copolymerization of
monomers comprising at least one .alpha.-hydroxy acid or derivative
thereof, including cyclic divesters and at least one
pseudo-.alpha.-amino acid. The .alpha.-hydroxy acids are generally
of the formula R.sub.1R.sub.2COHCO.sub.2H, where the R.sub.1 and
R.sub.2 groups are H, linear or branched alkyl groups. The
.alpha.-hydroxy acids may comprise a mixture of .alpha.-hydroxy
acids, at least one of the mixture of .alpha.-hydroxy acids having
R.sub.1 and R.sub.2 groups which are hydrogen and another
.alpha.-hydroxy acid having an R.sub.1 group which is CH.sub.3 and
R.sub.2 which is H. The pseudo-.alpha.-amino acids are generally of
the formula R.sub.5CHNHR.sub.6CO.sub.2H, where the R.sub.5 group is
a hydroxyl methyl or methyl thiol group and R.sub.6 is an amine
protecting group.
[0032] The amine protecting groups may be carbobenzyloxy, benzyl,
paramethoxybenzyl, benzyloxymethoxy, tert-butyloxycarbonyl or
[9-fluorenylmethyloxy]carbonyl.
[0033] The .alpha.-hydroxy acids are generally selected from
L-lactic acid, D,L-lactic acid, glycolic acid, hydroxy valeric acid
and hydroxybutyric acid. The at least one pseudo-.alpha.-amino acid
may be serine.
[0034] In a preferred aspect of the invention, the polymers are
poly-D,L-lactide-co-glycolide-co-pseudo-Z-serine ester (PLGpZS) and
poly-P,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS).
[0035] The polymers may contain biologically active moieties, such
as cell bioadhesion groups, macrophage stimulators, polyethylene
glycol, poly amino acids and/or protected amino acid residues,
covalently bound to the polymer directly or through side
groups.
[0036] In the preferred embodiment, the bioactive substituents are
linked to the polymer via the amino groups on the amino acid
moieties directly or via a suitable spacer molecule. The spacer
molecule can be selected from .alpha.-hydroxy acids represented by
the formula R.sub.7R.sub.8COHCO.sub.2H, where R.sub.7 or R.sub.8
groups are independently selected from H, linear or branched alkyl
units and .alpha.-amino acids represented by the formula
R.sub.9CHNHR.sub.10CO.sub.- 2H, where the R.sub.9 group is a
hydroxyl methyl or methyl thiol group and R.sub.10 is an amine
protecting group.
[0037] In accordance with a further aspect of the invention, the
invention provides a method of making a biodegradable,
biocompatible polyester, which comprises co-polymerizing at least
one .alpha.-hydroxy acid and at least one pseudo-.alpha.-amino
acid.
[0038] In accordance with another aspect of the present invention,
there is provided a process for making a biodegradable,
biocompatible polymer of the general formula provided herein which
comprises forming a mixture of monomers comprising at least one
.alpha.-hydroxy acid and at least one pseudo-.alpha.-amino acid
with an organic solvent solution of an esterification catalyst
under inert atmospheric conditions, copolymerizing the monomers and
isolating the resultant polymer. The catalyst used is preferably
stannous 2-ethylhexanoate.
[0039] The polymer formed by the process can be further deprotected
by solid phase catalytic reduction or alternatively by acid
catalysis using hydrogen bromide in acetic acid solution.
[0040] The process can also further comprise forming the polymer
into a film or microparticles.
[0041] In accordance with another aspect of this invention, there
is provided a particulate carrier for the delivery of biologically
active materials to a host, the carrier comprising a polymer,
including those having a molecular weight of about 5,000 to about
40,000 daltons, having the general formula: 2
[0042] wherein;
[0043] R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are selected
independently and are selected from H, linear or branched alkyl
groups;
[0044] R.sub.6 is selected from H, an amine protecting group, a
spacer molecule or a biologically active species;
[0045] X is selected from an O or S group; and
[0046] x and y are integers, including values such that at least
about 95% of the polymer is comprised of .alpha.-hydroxy acid
residues.
[0047] The particulate carrier generally has a particle size of
about 1 to 10 .mu.M.
[0048] In a further aspect of the present invention is a process
for making a particulate carrier for the delivery of at least one
biologically active material to a host, the process comprising:
[0049] (a) mixing an organic solvent phase comprising an
.alpha.-hydroxy acid polymer or copolymer with an aqueous
composition comprising dispersed or dissolved biologically active
material to form a first water-in-oil emulsion;
[0050] (b) dispersing the first water-in-oil emulsion into an
aqueous detergent phase to form a second water-in-oil-in-water
double emulsion;
[0051] (C) removing water from the second double emulsion to form
microspheres; and
[0052] (d) collecting the microspheres and having the biological
material entrapped therein.
[0053] The particulate carrier of the present invention can be used
as a composition having a biologically active material mixed
therewith or entrapped within. The biological materials used may be
selected from those which elicit an immune response. Such materials
may comprise Haemophilus influenzae proteins, such as a
non-proteolytic Hin-47 analog, D15, P1, P2, and P6. The
biologically-active material may comprise at least one influenza
virus, which may be a multivalent or monovalent influenza virus
vaccine, or influenza virus protein, such as an influenza virus
monovalent protein vaccine. In addition, the biologically-active
material may comprise at least one Moraxella catarrhalis protein,
such as the Tbp2 protein of M. catarrhalis. A further
biologically-active material which may be employed may be at least
one Helicobacter pylori protein, such as Urease. Other biological
material may include proteins, protein mimetics, bacteria,
bacterial lysates, viruses (e.g. respiratory syncytial virus),
virus infected cell lysates, DNA plasmids, antisense RNA, peptides
(e.g. CLTB-36 and M2), antigens, antibodies, pharmacological
agents, antibiotics, carbohydrates, lipids, lipidated amino acids
(e.g. tripalmitoyl cysteine), glycolipids, haptens and combinations
and mixtures thereof.
[0054] The first water-in-oil emulsion may additionally comprise at
least one organic solvent soluble adjuvant, which may be
lipophilic. Such organic solvent adjuvant may be selected from the
group consisting of BAY R1-005, tripalmitoyl cysteine and DC-chol.
The presence of a lipophilic moiety serves to increase the
encapsulation efficiency and to protect the antigen during
formulation and release and enables the particles to present
antigen to the immune system more efficiently than traditional
formulation and hence provides a more efficacious vaccine.
[0055] The first water-in-oil emulsion also may additionally
comprise at least one water soluble adjuvant, which may be a
polymeric water soluble adjuvant, such as PCPP or a mucosal
adjuvant, such as CT-X or subunit thereof or LT. The presence of
the water soluble adjuvant serves to increase the encapsulation
efficiency of the process and protects the antigen during
formulation and release and prevents the antigen to the immune
system more efficiently than traditional microparticle formulation,
thereby providing a more efficancious vaccine.
[0056] The present invention also provides an immunogenic
composition comprising the particulate carrier provided herein and
a physiologically acceptable carrier therefor. The composition can
be administered mucosally or parenterally. The immune response is
an antibody response which is a local or serum antibody response.
In accordance with this aspect of the invention, there is provided
a controlled or delayed release vaccine preparation in stable
particulate form and a method of making such a vaccine preparation.
The particles are microspherical and contain a matrix of
biodegradable polymer and antigen(s) and/or antigen plus
co-adjuvant containing regions.
[0057] Advantages of the invention include:
[0058] (a) fully biodegradable and biocompatible microparticle
formulation;
[0059] (b) facilitated antigen presentation to the cells of the
immune system resulting in improved antigen immunogenicity;
[0060] (c) improved formulating conditions which increase the
bioavailability of the antigen.
[0061] Additional embodiments of the present invention include the
use of the particulate carrier in diagnostic assays and for
therapeutic strategies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] The present invention will be further understood from the
following description with reference to the drawings, in which:
[0063] FIG. 1 is a schematic showing the ring opening
polymerization (ROP) of lactic acid dimer with glycolic acid dimer
and N-(carbobenzyloxy)-L-se- rine lactone with subsequent
deprotection in accordance with a preferred aspect of the present
invention.
[0064] FIG. 2 is a schematic showing the attachment of biologically
active moieties to polymer through the side chain of the
.alpha.-amino acid sub-unit within the polymer. Representative
targeting groups include polyethylene glycol (PEG) for water
solubility and circulation, macrophage stimulators and cell
bioadhesion groups. Spacer ligands derived from .alpha.-hydroxy
acids or .alpha.-amino acids may be incorporated to facilitate
attachment of the bioactive ligand.
[0065] FIG. 3 is a schematic detailing the process used to produce
microparticles in accordance with one embodiment of the invention.
In this figure, Hin-47 is a non-proteolytic recombinant protein
analog derived from Haemophilus influenzae (as described in U.S.
Pat. No. 5,506,139), Flu X31 is influenza strain X31 or A-Texas,
rD-15 is recombinant protein derived from Haemophilus influenzae
(as described in WO 94/12641), PVA=poly vinyl alcohol. Flu(tri) is
trivalent flu, Tbp2 is Moraxella catarrhalis transferrin binding
protein 2 and rUrease is recombinant Helicobacter pylori
urease.
[0066] FIG. 4 shows a typical size distribution for
poly-D,L-lactide-co-glycolide-co-pseudo-(Z)-serine ester (PLGpZS)
and poly-D,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS)
microparticles when prepared in the presence of PBS or a typical
protein (non-proteolytic Hin-47 analog) as determined by laser
diffraction measurements.
[0067] FIG. 5 shows a scanning electron micrograph of
microparticles prepared from
poly-D,L-lactide-co-glycolide-co-pseudo-(Z)-serine ester (PLGpZS)
and poly-D,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS) in
the presence of phosphate buffered saline(PBS).
[0068] FIG. 6 shows a scanning electron micrograph of
microparticles prepared from
poly-D,L-lactide-co-glycolide-co-pseudo-(Z)-serine ester (PLGpZS)
and poly-D,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS) in
the presence of a typical antigen/PBS mixture such as
Hin-47/PBS.
[0069] FIG. 7A shows the in vitro release profile for,
non-proteolytic Hin-47 analog encapsulated within PLG, PLGpZS and
PLGpS microparticles over a three month period obtained from 14 mg
samples (typical core loadings range from 2.5 to 5.7 .mu.g
protein/mg of microparticles) that were incubated in PBS (pH 7.4)
and maintained at 37.degree. C.
[0070] FIG. 7B compares the % cumulative release of non-proteolytic
Hin-47 analog from PLG, PLGpZS and PLGpS microparticles over a
three month period obtained from .about.14 mg samples (typical core
loadings range from 2.5 to 5.7 .mu.g protein/mg of microparticles)
that were incubated in PBS (pH=7.4) at maintained and 37.degree.
C.
[0071] FIG. 8A shows the serum IgG responses in mice immunized
subcutaneously (S.C.) following various immunization protocols by
the 47 kDa membrane protein from Haemophilus influenzae
(non-proteolytic Hin-47 analog). Groups of 5 mice were immunized on
days 1 and 35 with 250 .mu.L of PBS, pH 7.4, containing either 0.2
or 0.6 .mu.g of non-proteolytic Hin-47 analog incorporated into
PLG, PLGpZS or PLGpS microparticles. Sera obtained on days +10,
+24, +35, +46 and +60 were evaluated for the presence of
anti-Hin-47 IgG antibodies using an enzyme-linked immunosorbent
assay (ELISA).
[0072] FIG. 8B shows the serum IgG responses in mice immunized
subcutaneously (S.C.) following various immunization protocols by
the 47 kDa membrane protein from Haemophilus influenzae
(non-proteolytic Hin-47 analog). Groups of 5 mice were immunized on
days 1 and 35 with 250 .mu.L of PBS, pH 7.4, containing either 0.8
or 2.5 .mu.g of non-proteolytic Hin-47 analog physically mixed with
PLG microparticles. Sera obtained on days +10, +24, +35, +46 and
+60 were evaluated for the presence of anti-Hin-47 IgG antibodies
using an enzyme-linked immunosorbent assay (ELISA).
[0073] FIG. 8C shows the serum IgG response subtype profile for
pooled bleeds obtained on days +35 and +60 from the study conducted
as described in FIG. 8A and BB.
[0074] FIG. 9A shows the IgG serum antibody responses in mice
immunized intragastrically (I.G.) by the 47 kDa membrane protein
from Haemophilus influenzae (non-proteolytic Hin-47 analog). Groups
of 5 mice were immunized on days 1, 7, 14 and 57 with 500 .mu.L of
PBS, pH 7.4, containing 4 .mu.g of Hin-47 analog incorporated into
PLG, PLGpZS or PLGpS microparticles or physically mixed with PLG,
PLGpZS or PLGpS microparticles. Sera obtained on days +13, +35, +56
and +78 were evaluated for the presence of anti-Hin-47 IgG
antibodies using an enzyme-linked immunosorbent assay (ELISA).
[0075] FIG. 9B shows the serum IgG response subtype profile for
pooled bleeds obtained on days +56 and +78 from the study conducted
as illustrated in FIG. 9A.
[0076] FIG. 9C shows the serum IgA response for the bleed obtained
on day +78 from the study conducted as illustrated in FIG. 9A.
[0077] FIG. 10A shows the IgG serum antibody responses in mice
immunized intranasally (I.N.) by a (1:1) cocktail of the 47 kDa
membrane protein from Haemophilus influenzae (non-proteolytic
Hin-47 analog) and the 115 kDa membrane protein from Haemophilus
influenzae (rD-15). Groups of 5 mice were immunized on days 1, 7,
14 and 57 with 25 .mu.L of PBS, pH 7.4, containing 4 .mu.g of
non-proteolytic Hin-47 analog incorporated into PLG, PLGpZS or
PLGpS microparticles or physically mixed with PLG, PLGpZS or PLGpS
microparticles. Sera obtained on days +13, +35, +56 and +78 were
evaluated for the presence of anti-Hin-47 IgG antibodies using an
enzyme-linked immunosorbent assay (ELISA).
[0078] FIG. 10B shows the serum IgG response subtype profile for
pooled bleeds obtained on days +56 and +78 from the study conducted
as described in FIG. 10A.
[0079] FIG. 10C shows the serum IgA response for the bleed obtained
on day +78 from the study conducted as described in FIG. 10A.
[0080] FIG. 10D shows the lung lavage IgG response obtained on day
+78 from the study conducted as described in FIG. 10A.
[0081] FIG. 10E shows the lung lavage sIgA response obtained on day
+78 from the study conducted as described in FIG. 10A.
[0082] FIG. 11 shows the anti-Flu X31 (i.e. influenza virus type A
strain X31) IgG serum antibody responses following various
immunization protocols. Groups of 6 mice were immunized
subcutaneously (S.C.) on day 1 with 250 .mu.L of PBS, pH 7.4,
containing 1.5 .mu.g of HA incorporated into PLG, PLGpZS or PLGpS
microparticles. Sera obtained on days +21 and +33 and were
evaluated for the presence of anti-Flu X-31 IgG antibodies using an
enzyme-linked immunosorbent assay (ELISA).
[0083] FIG. 12 shows the anti-Flu X31 (i.e. influenza virus type A
strain X31) IgG serum antibody responses following various
immunization protocols. Groups of 6 mice were immunized
intranasally (I.N.) on days 1 and 34 with 25 .mu.L of PBS, pH 7.4,
containing 1.5 .mu.g of HA incorporated into PLG, PLGpZS or PLGpS
microparticles. Sera obtained on days +21, +33, +57, +78 and +92
were evaluated for the presence of anti-Flu X-31 IgG antibodies
using an enzyme-linked immunosorbent assay (ELISA).
[0084] FIG. 13 shows the hemagglutination inhibition antibody assay
(i.e. influenza virus strain A-Texas) responses for pooled sera
(days +21 and +42 or +57) following a single dose subcutaneous
administration. Groups of 6 mice were immunized subcutaneously
(S.C.) On day 1 with 250 .mu.l of PBS, pH 7.4, containing either
0.35 .mu.g of HA or 3.5 .mu.g of HA incorporated into PLGpS
microparticles or 0.35 .mu.g of HA and .about.2 .mu.g of BAY R1-005
or 3.5 .mu.g of HA and .about.20 .mu.g of BAY R1-005 incorporated
into PLGpS microparticles or 0.35 .mu.g of HA or 3.5 .mu.g of HA
physically mixed with PLGpS microparticles or 0.35 .mu.g of HA and
2 .mu.g of BAY R1-005 or 3.5 .mu.g of HA and 20 .mu.g of BAY R1-005
physically mixed with PLGpS microparticles. Sera obtained on days
+21 and +42 were evaluated for the inhibition of hemagglutination
of erythrocytes.
[0085] FIGS. 14a to c show the anti-Flu (trivalent) IgG serum
antibody responses (for each influenza virus strain contained in
trivalent vaccine; A/Texas (FIG. 14a), A/Johannesburg (FIG. 14b)
and B/Harbin FIG. 14C)) following various immunization protocols.
Groups of 8 mice were immunized subcutaneously (S.C.) on day 1 with
250 .mu.L of PBS, pH 7.4, containing 2.35 .mu.g of total HA (about
1/3 specific HA from each strain) incorporated into PLGpS
microparticles or PLGpS microparticles formulated in the presence
of BAY R1-005, DC-Chol or PCPP. Sera obtained on days +21, +42 and
+57 and were evaluated for the presence of anti-Flu (trivalent) IgG
antibodies (A/Texas, A/Johannesburg and B/Harbin) using an
enzyme-linked immunosorbent assay (ELISA).
[0086] FIGS. 15a to c show the strain specific hemagglutination
inhibition antibody assay (i.e. A/Texas (FIG. 15a), A/Johannesburg
(FIG. 15b) and B/Harbin (FIG. 15c)) responses (days +21, +42 and
+57) following a single dose subcutaneous administration. Groups of
8 mice were immunized subcutaneously (S.C.) on day 1 with 250 .mu.L
of PBS, pH 7.4, containing either 2.35 .mu.g of total HA
incorporated into PLGpS microparticles or 2.35 .mu.g of total HA
into PLGpS microparticles coencapsulating BAY R1-005, DC-Chol or
PCPP. Sera obtained on days +21, +42 or +57 were evaluated for the
inhibition of hemagglutination of erythrocytes.
[0087] FIG. 16 shows the results of a protection study performed on
mice immunized with a single dose of PLGpS/Flu (trivalent)
microparticles or PLGpS/Flu (trivalent) coencapsulating BAY R1-005,
DC-Chol or PCPP. The mice were challenged with homologous live
virus and monitored for weight changes and survival over a 2 week
interval.
[0088] FIG. 17a shows the serum IgG antibody responses in mice
immunized subcutaneously (S.C.) following various immunization
protocols by the transferrin binding protein from Moraxella
catarrhallis (Tbp-2). Groups of 5 mice were immunized on days 1, 28
and 43 with 250 .mu.L of PBS pH 7.4, containing 0.3 .mu.g of tbp-2
incorporated into PLGpS microparticles, physically mixed PLGpS
microparticles or formulated with Alum. Sera obtained on days +14,
+27, +42 and +55 were evaluated for the presence of anti-tbp-2 IgG
antibodies using an enzyme-linked immunosorbent assay (ELISA).
[0089] FIG. 17b shows the serum IgG antibody subtype response
profile for pooled bleeds obtained on day 55 from the study
conducted as described in FIG. 17a.
[0090] FIG. 18 shows the IgG serum antibody responses in mice
immunized intranasally (I.N.) with the transferrin binding protein
from Moraxella catarrhallis (Tbp-2). Groups of 5 mice were
immunized on days 1, 28 and 43 with either 10 .mu.L or 50 .mu.L of
PBS, pH 7.4, containing 6 .mu.g of Tbp-2 incorporated into
microparticles or physically mixed with PLGpS microparticles. Sera
obtained on days +14, +27, +42 and +55 were evaluated for the
presence of anti-Tbp-2 IgG antibodies using an enzyme-linked
immunosorbent assay (ELISA).
[0091] FIG. 19 shows the serum IgG antibody responses in guinea
pigs immunized parenterally following various immunization
protocols by the transferrin binding protein from Moraxella
catarrhallis (Tbp-2). Groups of 2 guinea pigs were immunized
intramuscularly with 5 .mu.g of Tbp-2 formulated with 400 .mu.L of
CFA on day 1 followed subcutaneously with 5 .mu.g of Tbp-2
formulated in 500 .mu.L of IFA on days 14 and 28, 5 .mu.g of Tbp-2
formulated with 500 .mu.L of Alum on days 1, 14 and 28 or 5.0 .mu.g
of Tbp-2 incorporated into PLGpS microparticles on days 1 and 28.
Sera obtained on days +40 and +56 were evaluated for the presence
of anti-tbp-2 IgG antibodies using an enzyme-linked immunosorbent
assay (ELISA).
[0092] FIG. 20 shows the serum IgG antibody subtype responses in
mice immunized subcutaneously (S.C.) or orally (I.G.) following
various immunization protocols by the recombinant protein rUrease
from Helicobacter pylori. Groups of 8 mice were immunized on days
1, 28 and 56 with 250 .mu.L of PBS pH 7.4, containing 10.0 .mu.g
(S.C.) or 40.0 .mu.g (I.G.) of rUrease incorporated into PLGpS
microparticles or 10.0 .mu.g (S.C.) or 40.0 .mu.g (I.G.) of rUrease
formulated in the presence of DC-Chol, CT-X, PCPP or LT
incorporated into PLGpS microparticles. Sera was obtained on day
+85 and were evaluated for the presence of anti-rUrease IgG
antibodies using an enzyme-linked immunosorbent assay (ELISA).
[0093] FIGS. 21a to b show the results of a protection study for
the mice described in FIG. 20 one month after challenge on day 85.
rUrease activity (for the mice immunized by subcutaneous or oral
routes) was measured in 1/4 of a whole stomach (antrum+corpus) 24
hours after the mice were killed.
DETAILED DESCRIPTION OF THE INVENTION
[0094] The novel polymers of the present invention are
biocompatible, degradable to benign metabolites which may be
present in the body and may possess biologically active moieties,
such as cell bioadhesion groups, macrophage stimulators, poly amino
acids and polyethylene glycol coupled to the polymer via at least
one spacer molecule selected from .alpha.-hydroxy acids and
.alpha.-amino acids. As such, the novel polymers possess
functionality.
[0095] Methods are also described for the synthesis of polymers
having advantageous properties for processing into microparticles
containing biologically active materials and for which chemical
modification with biologically active targeting groups is
possible.
[0096] In the preferred embodiments, the copolymers are produced by
the polymerization of .alpha.-hydroxy acids with
pseudo-.alpha.-amino acids and terpolymers produced by the
polymerization of two .alpha.-hydroxy acids with
pseudo-.alpha.-amino acids. The copolymer or terpolymer may then be
derivatised with biologically active targeting ligands via the
amino acid subunit by covalently coupling with the free amino group
directly or subsequent to further derivatization with a suitable
spacer ligand.
[0097] Amino Acid Monomer Synthesis
[0098] In general, an N-protected serine (or cysteine) is cyclized
via a Mitsunobu reaction (ref. 5) to give a four membered lactone
(or thiolactone).
[0099] This transformation gives rise to an ester (or thioester)
linkage. It is important to have protection on the amine portion of
the amino acid precursor that is compatible with the reaction
conditions. Preferentially the carbobenzyloxy (CBZ or Z) group is
used although other suitable functionalities, such as benzyl (Bn),
para-methoxybenzyl (MeOBn), benzyloxymethoxy (BOM),
tert-butyloxycarbonyl (t-BOC) or [9-fluorenylmethyl)oxy]carbonyl
(FMOC) may be employed.
[0100] The synthesis of the N-Z-L-Serine .beta.-Lactone monomer was
based on a modified procedure from the literature (ref. 6).
[0101] Copolymerization of .alpha.-Hydroxy Acid and Amino Acid
Containing Monomers and Functionalization of Amino Acid
Sidechains
[0102] Two methods are applicable for copolymerization of
.alpha.-hydroxy acid monomers. Polymerization via polycondensation
or from the melt (bulk polymerization) are possible
alternatives.
[0103] It has been long known that condensation polymerizations
are, problematic as relatively low molecular weight materials often
result with competing side reactions commonly giving rise to
unwanted byproducts (refs. 7 and 8).
[0104] However ring opening polymerization (ROP) of the cyclic
dimers of .alpha.-hydroxy acids, such as glycolide and lactide,
from the bulk phase was shown to proceed readily in the presence of
a variety of catalysts to give polymers of high molecular weights
with stannous octoate being preferred (refs. 9 to 15).
[0105] There are numerous methods for preparing poly(amino acids)
(refs. 16, 17 and 18) or pseudopoly (amino acids) (refs. 6 and
19).
[0106] The noted biodegradable properties of poly-.alpha.-hydroxy
acids (in particular those of 50:50 D,L-lactide and glycolide) and
poly(amino acids) has resulted in increased efforts to develop
methods for incorporating amino acids into the backbone of
.alpha.-hydroxy acid polymers (refs. 20 to 25).
[0107] Advances have been made in producing copolyesteramides
containing .alpha.-hydroxy acid sub-units, such as lactide or
glycolide, and .alpha.-amino acid sub-units, such as glycine or
lysine (refs. 22, 23 and 26).
[0108] The degradation rate of the biodegradable polymer and the
release rates of encapsulated materials from homopolymers of
glycolide, lactide or from copolymers of these materials has been
shown to be strongly influenced by their molecular weight and
structure, such as degree of crystallinity and relative
hydrophobicity or hydrophilicity. Specifically, microspheres
formulated from higher molecular weight polymers derived from
.alpha.-hydroxy acids degrade over longer periods of time than
lower molecular weight analogs. Similarly, highly crystalline
materials erode at rates much slower than amorphous analogs. This
is related to the accessibility of water to the hydrolytically
unstable ester linkages (ref. 27).
[0109] It has been established that random amorphous copolymers
composed of 50% D,L-lactide and 50% glycolide exhibit the most
advanced degradation rates (refs. 2 and 3) with 50% by weight
remaining after approximately 6 weeks, when immersed in PBS buffer
(pH=7.4).
[0110] The copolyesteramides described above are semi-crystalline
materials which may suffer from prolonged retention at the site of
administration long after the encapsulated materials are fully
released.
[0111] Since it would be advantageous to have a polymer that has
degraded at or near the point when the encapsulated material has
been fully released, we developed methods for randomly
incorporating equal amounts of D,L-lactide and glycolide into a
terpolymer which also contained pseudo-.alpha.-amino acid
sub-units. A terpolymer of relatively moderate molecular weight was
used to ensure the amorphous terpolymer would retain sufficient
mechanical strength for processing into films and microparticles
yet exhibit satisfactory polymer degradation and release rates for
entrapped materials.
[0112] The N-protected-L-serine lactone contains an ester bond
which may be polymerized via transesterification catalysts (ref.
6). Additionally it has been shown that six-membered ring lactones,
such as lactide and glycolide, can be copolymerized with
four-membered ring propiolactones by use of insertion/coordination
type catalysts/initiators (ref. 13). It was expected that efficient
transesterification catalysts, such as those derived from Sn
reagents, would be required if relatively sufficient reactivity of
all monomer units was to be achieved.
[0113] We used the copolymerization of glycolide, D,L-lactide and
N-Z-L-serine lactone mediated by stannous octoate. Deprotection of
the CBZ group of the copolymer or terpolymer can be achieved by
various methods. Solid phase catalytic reduction or acid catalysis
(ref. 27) are two possibilities (FIG. 1).
[0114] The resultant copolymer or terpolymer can be further
elaborated with targeting moieties such as cell adhesion epitopes,
poly ethylene glycol (PEG) ligands for circulation, macrophage
stimulators and poly amino acid grafts as depicted in FIG. 2. A
spacer unit may be incorporated, for example, an .alpha.-hydroxy
acid or a pseudo-.alpha.-amino acid unit, and may be readily
derivatised with the appropriate targeting units. The polymer so
formed has a molecular weight of from about 5,000 to about 40,000
daltons.
[0115] Microparticle Formation
[0116] The term "microparticle" as used herein refers to any
particulate carrier greater than 1 micron in size which is used for
the delivery of biologically active materials. The term
"microsphere" as used herein refers to a microparticle containing
one or more active ingredients (e.g. antigens, adjuvants, plasmid
DNA).
[0117] A flow diagram illustrating the process of microparticle
formation as described herein is shown in FIG. 3. In general, the
copolymer (PLG, PLGpZS or PLGpS) is solubilized solely or with
additional excipients present in a compatible solvent, such as
dichloromethane, ethyl acetate, acetone or mixtures thereof.
Excipients included in the formulation, such as sucrose, mannose,
trehalose or gelatin, serve as cryoprotectants or lyoprotectants.
Other materials possessing known adjuvancy, such as BAY R1-005
(BAY) (ref. 29) or tripalmitoyl cysteine (TPC) (ref. 30) or
3b[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol)
(ref. 31) may be included during formulation.
[0118] A 1% to 2% copolymer solution of total volume 12 mL is
preferably prepared. To this solution is added 800 .mu.L of
phosphate buffered saline (PBS) or 800 .mu.L of antigen solution
(concentration typically from 1 to 2 mg/mL) in PBS or other
stabilizing buffers which may contain additional excipients. Other
material possessing known adjuvancy, such as
poly[di(carboxylatophenoxy)-phosphazene] sodium salt, (Virus
Research Institute, Cambridge Mass.) (PCPP) or cholera toxin or
subunits thereof may be included during formulation. This mixture
is then homogenized to form a water in oil emulsion. Once formed,
this mixture is dispersed into 100 mL of a 0.5% to 10.0% aqueous
solution containing non-ionic emulsion stabilizers, such as poly
vinyl alcohol (PVA), methyl cellulose or Triton X-100. This mixture
is immediately homogenized to form a water in oil in water double
emulsion. The average size and polydispersity of the resultant
droplets can be conveniently measured through use of the Coulter
LS-100 light scattering detector. Typical size distributions when
PBS or Antigen/PBS mixtures are encapsulated range from 1 to 20
microns (with the majority less than 10 microns in size). The
solvent is then slowly removed via evaporation with gentle warming
to harden the incipient microspheres. Once the solvent is removed
the mixture is centrifuged to collect the microspheres and
repeatedly washed with deionized water to ensure complete removal
of residual emulsion stabilizers. The microspheres are then frozen
in a dry ice/acetone bath and lyophilized overnight to yield a
white freely flowing powder of microspheres (typically 1.0 to 12
microns in size as determined by light scattering measurements and
directly verified via scanning electron micrography).
[0119] The particle generally comprise a polymeric matrix having a
particle size of about 1 to about 50 .mu.M which comprises a
polymer having the biologically-active material entrapped therein,
with or without an adjuvant co-entrapped therein. The quantity of
biologically-active material and adjuvant which may be incorporated
into the polymeric matrix may vary widely and may comprise up to
about 50 wt % of the total particle mass.
[0120] It is clearly apparent to one skilled in the art, that the
various embodiments of the present invention have many applications
in the fields of medicine and in particular vaccination, diagnosis
and treatment of infections with pathogens including bacteria and
viruses. A further non-limiting discussion of such uses is
presented below.
[0121] Vaccine Preparation
[0122] In an embodiment, immunogenic compositions, suitable to be
used as, for example, vaccines, may be prepared from microparticles
as disclosed herein. The immunogenic composition containing a
biologically active immunogenic material can elicit an immune
response by the host to which it has been administered including
the production of antibodies by the host.
[0123] The immunogenic composition may be prepared as injectables,
as liquid solutions or emulsions. The microparticles may be mixed
with physiologically acceptable excipients which are compatible
with the microparticles. These may include, water, saline,
dextrose, glycerol, ethanol and combinations thereof. The vaccine
may further contain additional substances such as wetting or
emulsifying agents, pH buffering agents, or adjuvants to further
enhance the effectiveness of the vaccines. Vaccines may be
administered parenterally, by injection subcutaneously or
intramuscularly.
[0124] Alternatively, the immunogenic compositions comprising
microparticles formed according to the present invention may be
delivered in a manner to elicit an immune response at mucosal
surfaces. Thus, the immunogenic composition may be administered to
mucosal surfaces by nasal, oral (intragastric), buccal or rectal
routes. Oral formulations may include normally employed incipients,
such as pharmaceutical grades of saccharin, cellulose and magnesium
carbonate.
[0125] These compositions may take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders. In order to protect the microparticles and
the encapsulated material contained within the core of the
microparticle or which is physically mixed with the microparticles,
from gastric acidity when administered by the oral route, an acidic
neutralizing preparation (such as a sodium bicarbonate preparation)
is advantageously administered before, concomitant with or directly
after administration of the microparticles.
[0126] The vaccines are administered in a manner compatible with
the dosage formulation, and in such amount as to be therapeutically
effective, protective and immunogenic. The quantity to be
administered depends on the subject to be treated, including, for
example, the capacity of the subject's immune system to synthesize
antibodies, and if needed, to produce a cell-mediated immune
response. Precise amounts of microparticle and material having
biological activity required to be administered depend on the
judgement of the practitioner. However, suitable dosage ranges are
readily determinable by one skilled in the art and may be of the
order of micrograms to milligrams. Suitable regimes for initial
administration and booster doses are also variable, but may include
an initial administration followed by subsequent administrations.
The dosage of the vaccine may also depend on the route of
administration and thus vary from one host to another.
[0127] The polymers of the present invention as applied to vaccine
formulations, are also useful for new vaccine strategies, such as
with the use of DNA or antisense RNA. As microparticle carriers,
the polymers of the present invention can be prepared to contain
DNA coding for a gene or genes for an antigenic portion of a virus
such as the core protein or the envelope protein. As the DNA is
released from the carrier, the host cells take up the foreign DNA,
express the gene of interest and make the corresponding viral
protein inside the cell. Advantageously, the viral protein enters
the cells major histocompatibility complex pathway which evokes
cell-mediated immunity. Standard vaccine antigens in comparison,
are taken up into cells and processed through the MHC class II
system which stimulates antibody responses.
[0128] The DNA may be in the form of naked DNA which is free of all
associated proteins and which does not require a complex vector
system. The desired gene may be inserted into a vector, such as a
plasmid, encapsulated within the microparticle herein described,
and injected into the tissues of a mammal which expresses the gene
product and evokes an immune response.
[0129] Antisense oligonucleotides can also be used in conjunction
with the polymers of the present invention. Nucleic acid sequences
may be designed to bind to a specific corresponding mRNA sequence
for a known protein to inhibit the production of the protein. The
microparticles described herein, can be used to deliver such
antisense nucleotides (oligonucleotides or expressed nucleotides)
as a therapeutic strategy for the treatment of various
immunological diseases as well as hypertension, cardiovascular
disease and cancer.
[0130] The slow-release characteristic of the polymer
microparticles developed herein also has use in the field of
pharmacology where the microparticles can be used to deliver
pharmacological agents in a slow and continual manner. A wide range
of drugs, such as anti-hypertensives, analgesics, steroids and
antibiotics, can be used in accordance with the present invention
to provide a slow release drug delivery system.
[0131] The polymer in the form of a film having a biological agent
entrapped or physically admixed thereto, may also have use as a
coating for surgical implants and devices. For example, the polymer
as a film having antibiotic incorporated therein can be used to
coat surgically implanted catheters in order to provide continual
slow-release of antibiotics to combat infection.
[0132] The microparticle carrier may also be useful as a diagnostic
agent. Together with the appropriate antibody, imaging agents can
be incorporated with the microparticles. In this manner diseased
tissues can be targeted and imaged in order to identify or monitor
the clinical course of a disease.
[0133] The polymers, as microparticles, also have use in diagnostic
kits when used in conjunction with appropriate antibodies.
EXAMPLES
[0134] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific Examples. These Examples are
described solely for purposes of illustration and are not intended
to limit the scope of the invention. Changes in the form and
substitution of equivalents are contemplated as circumstances may
suggest or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive sense and
not for purposes of limitations.
[0135] Methods of chemistry, organic chemistry, polymer chemistry,
protein biochemistry and immunology used but not explicitly
described in this disclosure and these Examples are amply reported
in the scientific literature and are well within the ability of
those skilled in the art.
Example 1
[0136] This Example illustrates the preparation of N-Z-L-Serine
.beta.-Lactone.
[0137] The preparation of this cyclic N-protected amino acid
lactone was based on a modified procedure in which an
N-protected-.alpha.-amino acid is reacted to yield cyclized
pseudo-.alpha.-amino acid monomer (ref. 6). All glassware was
pre-dried overnight in an oven set at 120.degree. C. Prior to use
it was cooled in a vacuum desiccator and purged under a stream of
dry nitrogen for 10 minutes.
[0138] To a 1 L three necked round bottomed flask under nitrogen
was added triphenylphosphine (TPP; Aldrich; 7.87 mL; 50 mmol; FW:
174.16). To this was added 200 mL of anhydrous acetonitrile
(CH.sub.3CN; Aldrich): anhydrous tetrahydrofuran (THF; Aldrich)
solution (volume ratio 85:15) via syringe and stirred until the
solid TPP was dissolved. To this solution diethyl azodicarboxylate
(DEAD; Aldrich; 7.87 mL; 50 mmol; FW: 262.29) was added via syringe
and the solution stirred at room temperature for 30 minutes. The
solution was then cooled to about -45.degree. C. to -48.degree. C.
by immersing the reaction vessel in an acetonitrile/dry ice bath.
Once the internal temperature of the solution reached about
-45.degree. C., a solution of N-Carbobenzyloxy-L-Serine
(N-CBZ-L-Serine; Sigma; 11.94 g; 49.8 mmol; FW: 239.2) in 200 mL of
anhydrous CH.sub.3CN:THF (volume ratio 85:15) was slowly added via
dropping funnel over a period of 1 hour. The temperature of the
solution was maintained at about -45.degree. C. during the addition
and allowed to slowly warm to room temperature once the addition
was complete with continuous stirring overnight. This reaction
results in the formation of an ester bond between the serine
hydroxyl side chain and the carboxylic acid in the presence of the
CBZ protected .alpha.-amino group. Upon completion of the reaction
the solvents were removed via evaporation (35.degree. C. to
45.degree. C.). This yields a yellow oil/slurry (.about.35 g). To
this slurry was added 50 mL of dichloromethane:ethyl acetate
(volume ratio 85:15) solution which results in the precipitation of
1,2-dicarbethoxyhydrazine byproduct. This material was removed by
filtration under vacuum followed by solvent removal via
evaporation. The above procedure can be repeated to further remove
residual byproducts. The waxy solid crude material was then
purified via silica gel column chromatography with eluent. 85:15
dichloromethane:ethyl acetate as solvent. The product serine
lactone can be identified via thin layer chromatography as this
material has an R.sub.f of 0.75 on silica plates eluted with 85:15
dichloromethane:ethyl acetate when stained with a 1M
H.sub.2SO.sub.4 solution and is also UV visible. The product was
recrystallized from ethyl acetate:hexane (.about.1 L), filtered and
dried in vacuo.
[0139] A clean white solid is obtained in 40% yield after
recrystallization with a melting point (Tm=133-134.degree. C.) and
all other physical parameters (NMR, IR, mass spectroscopy,
elemental analysis) conforming to that previously demonstrated
(ref. 6).
Example 2
[0140] This Example illustrates the preparation of the copolymer
poly-D,L-Lactide-co-Glycolide-co-pseudo-Z-Serine Ester (PLGpZS) as
shown in FIG. 1.
[0141] Glassware was pre-dried overnight. Prior to use it was
cooled in a vacuum desiccator. Additionally the polymerization
vessel (glass ampule) must be siliconized (SurfaSil; Pierce; 2%
solution in toluene) and all transfer reactions and additions of
reagents and monomers to polymerization vessel must be conducted in
a glove box maintained under a dry nitrogen environment.
[0142] Prior to polymerization the D,L-lactide
(2,6-dimethyl-1,4-dioxane-2- ,5-dione; Aldrich; FW: 144.13) and
glycolide (Boehringer Ingelheim; FW: 116.096) was recrystallized
from anhydrous ethyl acetate in the glove box and dried in vacuo
for about 2 days. Once fully dried the monomers can be stored in
the glove box with the freshly recrystallized serine lactone
(stored at 0.degree. C.) of Example 1 brought directly into the
glove box. All monomers and catalyst/initiators were weighed and
transferred to glass ampules within the glove box.
[0143] The total combined mass of monomer transferred to the
ampoule typically ranges from 1 g to 5 g with the molar ratio of
D,L-lactide:glycolide:serine lactone ranging from 42.5:42.5:15.0 to
49.0:49.0:2.0. A molar ratio of D,L-lactide:glycolide:serine
lactone, of 47.5:47.5:5.0 was used in the preferred embodiment. A
stock solution of catalyst (stannous 2-ethylhexanoate
(Sn(Oct).sub.2; Sigma; FW 405.1, 1.25 g/mL) in anhydrous chloroform
(Aldrich) was prepared in the glove box and added via microsyringe
to the glass ampule (molar ratio of catalyst to monomer=1/1000). A
stir bar was also placed in the ampule. A greased ground glass
joint valve was placed on the ampule to preserve the inert
environment during removal from the glove box. The ampule was then
directly placed on a vacuum line with slow removal of chloroform by
evaporation. The ampules were then placed in an oil bath at ca.
120.degree. C. to bring all reagents into the melt followed by
flame sealing and placement in a thermoregulated oven at
120.degree. C. for 28 hours. After reaction the ampules are
quenched by placing in liquid nitrogen and stored at -20.degree. C.
until further work up. The ampule was cracked and crude polymer
recovered by dissolving in chloroform. The solvent was removed by
evaporation and crude polymer dried in vacuo to give an amber
crystalline material (yield 80% to 90%).
[0144] The polymer was purified by dissolving in chloroform,
filtering off insoluble material and precipitating into hexane
(Aldrich). The polymer was recovered by filtration and this
procedure was repeated to ensure the complete removal of unreacted
monomer. The polymer was dried in vacuo for about 2 days to give a
clean white powder in 30% to 35% overall yield after second
precipitation. The molecular weight of this material was dependent
on reaction time with typical values of Mw=17,000-22,000,
Mn=6,500-8,000. Differential scanning calorimetry (DSC) analysis
indicates a single transition indicative of a random amorphous
polymer. Glass transitions (Tg) range from 39.degree. C. to
43.degree. C. dependent on the molecular weight of the material
obtained. The serine content of the polymer was determined by amino
acid analysis (AAA) diagnostic for the phenylthio isocyanate serine
derivative obtained by hydrolysis of the polymer, .sup.1H NMR
(integration of aromatic residues of the CBZ protecting group on
serine relative to the glycolide and D,L-lactide sub-units) and
elemental analysis (nitrogen present only in the side chain of
serine). The AAA analysis typically indicated 1.7% to 2.1% serine
content with the .sup.1H NMR analysis indicating 2.0% to 2.5% and
the elemental analysis indicating 2.4% to 3.4% serine respectively.
IR analysis of the polymer was diagnostic for the presence of
ester, carbamate and hydroxyl groups.
[0145] .sup.1H NMR also allowed for the determination of the
relative incorporation efficiencies of all monomer components under
the stated reaction conditions. Typical ratios of
D,L-lactide:glycolide:Z-serine found in purified polymer are
reproducibly 52.0% to 54.0% D,L-lactide, 41.0% to 43.5% glycolide
and 2.0% to 2.5% Z-serine respectively.
[0146] .sup.1H NMR and .sup.13C NMR signal intensities for
resonances unique to glycolide or D,L-lactide are well resolved
from each other and sensitive to sequence effects. From the
observed patterns a random sequence distribution is supported.
Example 3
[0147] This Example illustrates the preparation of the copolymer
poly-D,L-Lactide-co-Glycolide-co-pseudo-Serine Ester (PLGpS) as
shown in FIG. 1.
[0148] All glassware was pre-dried overnight. Prior to use it was
cooled in a vacuum desiccator and purged under a stream of dry
nitrogen for 10 minutes. All reactions were conducted under inert
atmosphere of dry nitrogen.
[0149] To a 2 necked 100 mL round bottomed flask equipped with a
stir bar was placed 400 mg of polymer (PLGpZS). To this was added a
10 mL solution of 30% hydrogen bromide in acetic acid (Aldrich; FW:
80.92) which was sufficient for slurry formation. The slurry was
stirred for 30 to 45 minutes and quenched by dropwise addition of
anhydrous diethyl ether (Aldrich) followed by anhydrous methanol
(Aldrich). This results in polymer precipitation which was then
isolated by vacuum filtration. The crude polymer precipitate was
washed with diethyl ether and reprecipitated from
chloroform:hexane. The purified polymer was dried in vacuo for
about 2 days to give a clean white powder in 50% to 60% overall
yield. The molecular weight ranged from Mw 15,000-18,000,
Mn=5,000-6,500. The rate of deprotection of the CBZ group is faster
than the competitive cleavage of the ester backbone with HBr/Acetic
acid. However, under these conditions there is broadening of the
molecular weight distribution and reduction in the molecular weight
of the product as a consequence of using this reagent. This trend
can be reduced by conducting the reaction for shorter time
intervals or eliminated by removing the protecting group via
hydrogenation using hydrogen in the presence of palladium on
charcoal. DSC analysis indicates a single transition indicative of
a random amorphous polymer. Glass transitions (Tg) range from
42.degree. C. to 45.degree. C. depending on the molecular weight of
the material obtained. The serine content of the polymer was
determined by amino acid analysis (AAA) diagnostic for the
phenylthio isocyanate serine derivative obtained by hydrolysis of
the polymer and elemental analysis (nitrogen present only in the
side chain of serine). The AAA analysis typically indicated 1.4% to
1.7% serine content and the elemental analysis indicating 2.0% to
2.7% serine respectively. IR analysis of the polymer was diagnostic
for the presence of ester, amine and hydroxyl groups.
[0150] .sup.1H NMR for residual protected polymer indicated that
greater than 90% of the N-carbobenzyloxy groups were successfully
removed. With shorter reaction times the extent of deprotection is
concomitantly reduced. Typical ratios of
D,L-lactide:glycolide:Z-serine:serine found in purified polymer are
reproducibly 53.0% to 55.0% D,L-lactide, 40.0% to 43.0% glycolide,
0.15% to 0.25% Z-serine and 1.7% to 2.1% serine respectively.
Example 4
[0151] This Example illustrates the production of a film from the
copolymers synthesized in Examples 2 and 3.
[0152] To produce the film, 50 mg of poly-D,L-Lactide-co-Glycolide
(PLG) (Mw=31,000), poly-D,L-Lactide-co-Glycolide-co-pseudo-Z-Serine
Ester (PLGpZS) (Mw=20,000) or
poly-D,L-Lactide-co-Glycolide-co-pseudo-Serine Ester (PLGpS)
(Mw=19,000) was weighed out and placed in a 10 mL beaker. Anhydrous
chloroform (1 mL) was added to dissolve the copolymer. This
solution was filtered and added dropwise to a microscope slide
placed in a petri dish. The petri dish was then covered with a 250
mL beaker to ensure slow evaporation over 48 hours. The resultant
films were translucent and contact angle measurements performed
using a goniometer gave average values of 75.degree. for PLG,
75.degree. for PLGpZS and 68.2.degree. for PLGpS respectively. Thus
the PLG and PLGpZS copolymers are of comparable hydrophobicity with
the PLGpS copolymer proving to be slightly more hydrophilic and of
higher surface energy.
Example 5
[0153] This Example illustrates the process of microparticle
formation encapsulating PBS or antigen/PBS (microsphere
formation).
[0154] A flow diagram illustrating the process of microparticle
formation as described herein is shown in FIG. 3.
[0155] Specifically, 100 mg of copolymer was added to 12 mL of
dichloromethane. To this was added 800 .mu.L of phosphate buffered
saline (PBS) solution or 800 .mu.L of non-proteolytic Hin-47 analog
(concentration typically from 1 to 2 mg/mL) in PBS. This mixture
was then homogenized (20 seconds at 6,000 rpm). Once formed this
mixture was dispersed into 100 mL of a 1.0% aqueous solution of
poly vinyl alcohol (PVA) and immediately homogenized (40 seconds at
8,000 rpm) to form a water in oil in water double emulsion. Typical
size distributions when PBS or a typical antigen (Hin-47/PBS) is
used as encapsulant are depicted in FIG. 4. Polydisperse
microparticles (with the majority less than 10 microns in size)
were formed under these conditions. The solvent was then slowly
removed via evaporation and the microspheres collected by
centrifugation. The particles were washed (5.times.) with deionized
water and then frozen in a dry ice/acetone bath and lyophilized
overnight to yield a white freely flowing powder of microspheres
(typically 1.5 to 10 microns in size as determined by light
scattering measurements and directly verified via scanning electron
micrography). A representative scanning electron micrograph for
PLGpZS or PLGpS microspheres encapsulating PBS is shown in FIG. 5.
A representative scanning electron micrograph for PLGpZS or PLGpS
microspheres encapsulating a typical antigen (non-proteolytic
Hin-47 analog) in PBS is shown in FIG. 6.
[0156] By the method stated above microparticles containing several
different antigen(s) and/or antigen(s)+adjuvant have been prepared
(see Tables 1 and 5).
Example 6
[0157] This Example illustrates the microparticle core loading
efficiency and antigen epitope recovery from such
microparticles.
[0158] Two variations of the same method were employed to determine
the antigen content or "core loading" of the microparticles
isolated. Amino acid analysis was performed on the hydrosylates of
microparticles obtained by either acid hydrolysis (6M HCl) of the
solid particles or by base/SDS hydrolysis (0.1N NaOH/1% SDS)
followed by neutralization with 0.1N HCl. Alternatively, the solid
microspheres can be dissolved in DMSO a compatible solvent
solubilizing both polymer and protein and amino acid analysis
performed directly on the lyophilized sample. The acid or base
mediated hydrolysis proved to be the preferred method giving the
most reproducible results (+/-5%). Where available a validated
Enzyme Linked Immunosorbant Assay (ELISA) polyclonal assay was
performed on the hydrosylates to determine the epitope
equivalence.
[0159] Specifically, for the quantitation of non-proteolytic Hin-47
analog antigen by ELISA the non-proteolytic Hin-47 analog antigen
was captured on affinity purified guinea pig anti-Hin47 coated
microtitre wells (Add 50 .mu.L of a 2 .mu.g/mL solution of
non-proteolytic Hin-47 analog antigen per well), which have been
blocked with 5% skim milk in PBS. The antigen present was detected
by an affinity purified rabbit anti-Hin-47 followed by horse radish
peroxidase F(abs)2, donkey anti-rabbit IgG. To develop the color of
this reaction 100 .mu.L of the substrate H.sub.2O.sub.2 (9 parts)
in the presence of tetramethylbenzidine (TMB) (1 part), and the
reaction progress terminated by addition of 50 .mu.L of a 2M
sulphuric acid solution to each well. The intensity of the color
(read at 450 nm) was directly proportional to the amount of
non-proteolytic Hin-47 analog in the well. The concentration of
non-proteolytic Hin-47 analog in each test sample was derived by
extrapolation from a standard curve which was obtained by multiple
dilutions of a reference sample. All samples were analyzed in
duplicate.
[0160] The amino acid analysis for core loading and Hin-47 specific
polyclonal ELISA analysis of epitope recovery for microparticles
encapsulating non-proteolytic Hin-47 analog or non-proteolytic
Hin-47 analog plus BAY R1-005 within PLG, PLGpZS and PLGpS
copolymers is shown in Table 2. The core loadings for
microparticles prepared solely from Hin-47 range from 20% to 43%
with from 10% to 31% of the epitope preserved during formulation.
The core loadings for microparticles prepared from non-proteolytic
Hin-47 analog in the presence of BAY R1-005 range from 26% to 59%
(an absolute increase of 6% to 16%) with from 20% to 39% (an
absolute increase of 4% to -18%) of the epitope preserved during
formulation. Thus formulation in the presence of BAY R1-005
concomitantly increases the loading efficiency and serves to
protect the epitope.
[0161] The amino acid analysis of core loading for microparticles
encapsulating rD-0.15 within PLG, PLGpZS and PLGpS copolymers is
shown in Table 3. Core loadings ranging from 39% to 44% were
obtained. This protein was membrane derived and thus more
hydrophobic than the non-proteolytic Hin-47 analog of the previous
example. Increased encapsulation efficiencies relative to
non-proteolytic Hin-47 analog of Example 10 were routinely observed
when using this antigen.
[0162] The amino acid analysis for core loading and epitope
recovery for microparticles encapsulating a cocktail of
non-proteolytic Hin-47 analog and rD-15 (1:1 cocktail) within PLG,
PLGpZS and PLGpS copolymers is shown in Table 3. It was expected
that coencapsulation of non-proteolytic Hin-47 analog in the
presence of rD-15 would result in higher overall loading
efficiencies. Core loadings ranging from 35% to 62% were obtained
with from 8% to 26% of the non-proteolytic Hin-47 analog epitope
preserved during formulation. The overall loading efficiency of the
more hydrophilic component (non-proteolytic Hin-47 analog) was
increased by coencapsulation with rD-15 when formulated with PLGpZS
or PLGpS copolymers. This effect was not observed when PLG was
used. Irrespective of copolymer formulation similar non-proteolytic
Hin-47 analog epitope recovery as compared with Example 10 was
obtained.
[0163] The amino acid analysis of core loading for microparticles
encapsulating Flu X-31 or Flu X-31 and BAY R1-005 within PLG,
PLGpZS and PLGpS copolymers is shown in Table 4. Core loadings
ranging from 26% to 40% were obtained with Flu X-31 and for Flu
X-31 in the presence of BAY R1-005 core loadings ranging from 31%
to 53% were obtained. Thus, an absolute increase of 5% to 13% was
obtained when formulating in the presence of BAY R1-005. The only
exception was the formulation of Flu X-31 with BAY R1-005 and PLGpS
where an absolute decrease of 9% was observed.
[0164] A core loading of 21% for PLGpS microparticles encapsulating
Flu A-Texas was obtained. For PLGpS microparticles encapsulating
Flu A-Texas in the presence of BAY R1-005 a core loading of 32% was
obtained. When a different strain of Flu was employed (A-Texas)
with BAY R1-005 and PLGpS copolymer the tendency to increase the
encapsulation efficiency was once again observed. When formulating
PLGpS with Flu A-Texas in the presence of BAY R1-005 an absolute
increase of 11% was obtained. This suggests that the formulation
for PLGpS and proteins in the presence of BAY R1-005 must be
optimized in each case. As in Example 10, the addition of the
coadjuvant BAY R1-005 has resulted in higher loading
efficiencies.
[0165] The amino acid analysis of core loading, for microparticles
encapsulating Flu (trivalent) vaccine or Flu (trivalent) vaccine
and BAY R1-005, DC-Chol or PCPP within PLGpS copolymers is shown in
Table 6. Excellent core loading efficiencies ranging from 72% to
104% were obtained using the procedure of Example 5. The
improvement in encapsulation efficiencies is a general result
observed when the optimum concentration of protein is used to form
the primary emulsion. In this case, the optimum concentration was
experimentally determined to be about 0.5 mg/mL.
[0166] The amino acid analysis for core loading and rUrease
specific polyclonal ELISA analysis of epitope recovery for
microparticles encapsulating rUrease or rUrease plus DC-Chol, PCPP
or CT-X within copolymers is shown in Table 7. Epitope recovery is
defined as the difference between the total protein obtained by
amino acid analysis and total protein obtained by polyclonal ELISA
expressed as a percentage. The core loadings for microparticles
prepared solely from rUrease is about 46% with about 72% of the
epitope recovered during formulation. Similarly the core loadings
for microparticles prepared from rUrease in the presence of DC-Chol
is about 44% with about 69% epitope recovered. Slightly higher
total protein was determined for microparticles prepared from
rUrease in the presence of PCPP. A core loading of 67% and epitope
recovery of about 55% was found for this combination. For the
rUrease/CT-X combination, the total core loading for rUrease could
not be estimated by amino acid analysis since both rUrease and CT-X
contribute to this value. A polyclonal ELISA assay for CT-X was
developed and microparticle hydrosylates assayed for total rUrease
and CT-X separately. An epitope recovery of 0.53% was found for
rUrease and 59% for CT-X respectively. SDS-PAGE and Western blots
performed on these microparticle hydrosylates confirmed the
presence of non-degraded rUrease and CT-X.
[0167] Suitable controls were performed to ensure that the
polyclonal ELISA assay for rUrease was unaffected by additives,
such as DC-Chol and PCPP. In the case of PCPP, the assay was often
problematic and variable. No similar problem was found when
assaying rUrease in the presence of DC-Chol.
Example 7
[0168] This Example illustrates the in vitro release rates and
total cumulative % recovery of protein for a model antigen
(non-proteolytic Hin-47 analog) encapsulated within PLG (used as a
control for comparison purposes) and synthesized copolymers PLGpZS
and PLGpS.
[0169] The PLG formulated into microspheres containing antigen was
determined to be of molecular weight; Mw=26,000 with a 50:50 ratio
of D,L-Lactide:Glycolide. This material is of similar constitution
and molecular weight to that obtained for copolymers PLGpZS and
PLGpS of Examples 2 and 3. In a typical experiment, 14 mg of
microspheres (core loading of non-proteolytic Hin-47 analog for
PLG=2.8 .mu.g/mg, PLGpZS=5.7 .mu.g/mg and PLGpS=2.5 .mu.g/mg as
determined by amino acid analysis of the microsphere hydrosylates)
was placed in a 2 mL eppendorf tube to which was added 1.0 mL of
PBS (pH=7.4). The tube was then placed in a thermoregulated oven
maintained at 37.degree. C. without agitation. At various time
intervals the PBS solution was extracted and analyzed for total
protein via amino acid analysis. After each extraction the PBS
solution was replaced with continued sampling to a maximum of 90
days. In control experiments, the majority of the microspheres
(>80% by weight) prepared from PLG or PLGpS copolymers were
consumed by day 45. The degradation of microspheres formulated from
PLGpZS copolymers was observed to be somewhat slower requiring 60
days to erode to essentially the same extent.
[0170] Similar antigen release trends were observed with each group
of microparticles wherein small amounts of protein were released
over the first few days. This diminished to near undetectable
limits up to day 14 whereafter the protein release rate steadily
increased to a maximum at about day 30. Subsequent to this time,
the release of protein fell to levels again approaching the limit
of detection. The total % cumulative recovery of protein from these
samples ranged from 40% to 65% relative to the respective core
loadings of each group of microspheres.
[0171] FIG. 7A illustrates the release rate at specific time points
for each sample and FIG. 7B shows the % cumulative release for each
sample examined. It is evident that under these conditions the best
recovery of protein from microspheres follows the order
PLGpS>PLG>PLGpZS although the core loading for the PLGpZS
microparticles was approximately twice that of the PLG or PLGpS
analogs and this may influence the release rate. In addition, this
matrix has been shown to degrade at a slower rate and thus there
may be residual material within the microparticles. Evidence for
this can be seen in FIG. 7B whereby a marginal increase in protein
recovery was observed from day 60 to day 90 for PLGpZS
microparticles. Over this same time interval, protein recovered
from PLG or PLGpS microparticles was essentially non-existent.
[0172] In control experiments, supernatant solutions of PBS
obtained from periodic extractions of PBS loaded PLG, PLGpZS or
PLGpS microparticles incubated at 37.degree. C. were monitored for
pH changes. It is known that the erosion process for PLGA matrices
is accompanied by changes in the pH microenvironment within the
microparticle (ref. 36). This can have a serious effect on protein
stability as pH induced conformational changes can ensue as a
consequence of these changes. An indication of the magnitude of
these changes can be obtained by monitoring the pH of the
surrounding medium. We have found that incorporation of small
percentages of amino acid sub-units within the copolymer can retard
this process. This may be of importance during the release phase
for proteins sensitive to acidic pH. Specifically when incubating
.about.10 mg of microparticles in 1.2 mL of PBS buffer (pH=7.4) it
required approximately 16 days for supernatant extractions of PLG
(Mw=26,000 daltons) to fall below pH 5.0. By analogy the pH of
supernatant extractions obtained from PLGpZS (Mw=20,000 daltons,
approximately 2.0% serine incorporated), or PLGpS (Mw=18,000
daltons, approximately 1.7% serine incorporated) was determined to
be approximately 5.5 to 6.2 respectively. The degradation rates for
PLG and PLGpS examined in this study were similar (as measured by
mass loss of matrix) whereas the PLGpS matrix degraded at a reduced
rate.
[0173] Thus the in vitro release study demonstrates that a single
dose delayed release delivery system can be achieved through use of
polymeric microparticles formulated from PLGpZS or PLGpS
copolymers. In addition as pH changes with matrix erosion can have
a deleterious effect on the protein stability it may be
advantageous to employ matrices derived from pseudo-serine ester
such as PLGpZS or PLGpS wherein there exists some buffering
capacity for these changes during the protein release phase.
Example 8
[0174] This Example illustrates the immunogenicity of
non-proteolytic Hin-47 analog encapsulated or physically mixed with
microparticles in mice which were immunized subcutaneously.
[0175] To examine the immunogenicity of non-proteolytic Hin-47
analog entrapped in PLG, PLGpZS and PLGpS microparticles formed in
accordance with the present invention, groups of five, 6 to 8 week
old female BALB/c mice (Charles River Breeding Laboratories,
Wilmington, Mass.) were immunized subcutaneously (S.C.) with the
following amounts of antigen in 250 .mu.L of PBS (pH 7.4) on days 1
and 35: PLG, PLGpZS and PLGpS microparticles prepared as described
in Example 5 containing 0.2 .mu.g or 0.6 .mu.g of non-proteolytic
Hin-47 analog (FIG. 8A); and PLG microparticles prepared as
described in Example 5 physically mixed with 0.8 .mu.g or 2.5 .mu.g
of non-proteolytic Hin-47 analog (FIG. 8B).
[0176] The mice showed no gross pathologies or behavioral changes
after receiving microparticles that contained encapsulated
non-proteolytic Hin-47 analog or microparticles that were
physically mixed with non-proteolytic Hin-47 analog. Sera were
obtained on days +10, +24, +35, +45 and +60 and were evaluated for
the presence of anti-Hin-47 IgG antibodies by antigen specific
ELISA. All samples were analyzed in duplicate. Microtiter plate
wells were incubated overnight at room temperature with 100 .mu.L
of 0.2 .mu.g/mL non-proteolytic Hin-47 analog in 0.05M
carbonate-bicarbonate buffer (pH 9.0). The plates were washed with
PBS+0.05% Tween 20 (operationally defined as washing buffer). Wells
were incubated with 200 .mu.L of 5% skim milk (operationally
defined as blocking buffer). After washing with PBS+0.05% Tween 20,
the plates were incubated for 1 h at 37.degree. C. with 100 .mu.L
of sample serially diluted in blocking buffer. Wells were washed
with PBS+0.05% Tween 20 and 100 .mu.L of HRP-conjugated antibody
(goat anti-mouse IgG (H+L) (Jackson), sheep anti-mouse IgG1
(Serotec), goat anti-mouse IgG2a (Caltag) or goat anti-mouse IgG2b
(Caltag) in blocking buffer was added to each well. After 1 hour
incubation at 37.degree. C., the wells were washed five times with
PBS+0.05% Tween 20 and 100 .mu.L of freshly prepared colorizing
substrate [H.sub.2O.sub.2 (9 parts) and TMB (1 part)] is added to
each well. After 5 minutes incubation in the dark at room
temperature the reaction is stopped by adding 50 .mu.L of a 2M
H.sub.2SO.sub.4 solution and the optical density of the fluid in
each well was determined at 450 nm using a microplate reader. A
normal mouse sera pool was used to establish baseline optical
density values in the assay. Hyperimmune mouse Hin-47 antiserum was
used as a positive control. Pre-immune sera was used as negative
control.
[0177] For serum IgA analysis the above procedure was conducted
with the following modification. Microtiter plate wells were
incubated overnight at room temperature with 100 .mu.L of 1.3
.mu.g/mL non-proteolytic Hin-47 analog in 0.05M
carbonate-bicarbonate buffer (pH=9.0), and 100 .mu.L of HRP
conjugated rabbit anti-mouse IgA (ZYMED, CA) at 0.06 .mu.g/mL was
added to each well.
[0178] For secretory IgA analysis, the above procedure was
conducted with the following modification. 100 .mu.L of HRP
conjugated rat anti-mouse IgA (Biotin-Pharmigen) at 0.06 .mu.g/mL
was added to each well.
[0179] The serum antibody titres following immunization are shown
in FIGS. 8A and 8B. The results of immunizations indicate that
antigen presented to the immune system entrapped in PLG, PLGpZS or
PLGpS microparticles (FIG. 8A) was substantially more immunogenic
than soluble antigen at doses sub-optimal to that required with
soluble antigen alone. In addition, no dose dependence was observed
with encapsulated non-proteolytic Hin-47 analog of dose 0.2 .mu.g
or 0.6 .mu.g, whereas a marginal increase in titre was found with
soluble non-proteolytic Hin-47 analog of dose 0.8 .mu.g or 2.5
.mu.g respectively.
[0180] The results of immunizations indicate that antigen presented
to the immune system when physically mixed with PLG microparticles
(FIG. 8B) is marginally more immunogenic than soluble antigen at
similar dose to that given with soluble antigen alone (0.8 .mu.g or
2.5 .mu.g). However, administration of antigen in soluble form or
admixed with microparticles elicits a response several orders of
magnitude less than that seen for antigen encapsulated within the
microparticles demonstrating the advantages of encapsulation over
soluble or physically mixing alone.
[0181] Interestingly, the IgG subtype profile (FIG. 8C) for pooled
serum obtained from the bleeds on day 35 and 60 for microparticle
encapsulated non-proteolytic Hin-47 analog, microparticles
physically mixed with non-proteolytic Hin-47 analog or soluble
non-proteolytic Hin-47 analog shows that by day 35 IgG1 was the
dominant subtype with some IgG2b detected irrespective of
formulation. By day 60 it was evident that the IgG subtypes induced
by non-proteolytic Hin-47 analog encapsulated within microparticles
had undergone class switching such that IgG1, IgG2a and IgG2b are
more equally represented. The IgG subtypes induced by
non-proteolytic Hin-47 analog physically mixed with particles or in
soluble form by day 60 was nominally the same as that determined
for day 35 with the IgG1 subtype dominant.
[0182] Thus, it can be concluded that the qualitative nature of the
immune response mediated by microparticles encapsulating antigen is
substantially different than that obtained by physically mixing
with microparticles or by soluble antigen alone. The presence of
IgG2a subtype in the BALB/c mouse model is generally accepted to be
indicative of a TH.sub.1 pathway, and IgG1 indicative of a TH.sub.2
pathway. It is evident that via the subcutaneous route the TH.sub.2
pathway is favored for soluble antigen or for antigen physically
mixed with microparticles, whereas with antigen encapsulated within
microparticles a more balanced TH.sub.1/TH.sub.2 response is
attainable.
Example 9
[0183] This Example illustrates the immunogenicity of
non-proteolytic Hin-47 analog entrapped in PLG, PLGpZS and PLGpS
microparticles formed in accordance with the present invention, in
mice immunized intragastrically.
[0184] Groups of five, 6 to 8 week old female BALB/c mice (Charles
River Breeding Laboratories, Wilmington, Mass.) were immunized
intragastrically (I.G.) with the following amounts of antigen in
250 .mu.L of 0.15 M NaHCO.sub.3 (pH 9.0) on days 1, 7, 14 and 57:
PLG, PLGpZS and PLGpS microparticles prepared as described in
Example 5 containing 4.0 .mu.g of non-proteolytic Hin-47 analog;
and PLG, PLGpZS and PLGpS microparticles prepared as described in
Example 5 physically mixed with 4.0 .mu.g non-proteolytic Hin-47
analog (FIG. 9A).
[0185] The mice showed no gross pathologies or behavioral changes
after receiving microparticles that contained encapsulated
non-proteolytic Hin-47 analog or microparticles that were
physically mixed with non-proteolytic Hin-47 analog. Sera were
obtained on days +13, +35, +56 and +78 and were evaluated for the
presence of anti-Hin-47 IgG and IgA antibodies by antigen specific
ELISA as described in Example 8.
[0186] Sera and intestinal washes were examined for the presence of
Hin-47-specific antibodies. To detect and quantify anti-Hin-47 IgG
and sIgA in the intestinal lumen, mice were sacrificed by cervical
dislocation, their small intestines removed and examined for the
presence of antigen-specific antibodies. Individual small
intestines were detached from the pyloric sphincter to the caecum
and everted over capillary tubes. The everted intestines were
incubated in 5 mL of ice cold enzyme inhibitor solution (0.15 M
NaCl, 0.01 M Na.sub.2HPO.sub.4, 0.005 M EDTA, 0.002 M PMSF, 0.05
U/mL Aprotinin, and 0.02% v/v NaN.sub.3) for 4 hours. Intestines
were removed and the supernatants clarified by centrifugation
(1000.times.g, 20 minutes) and stored at 0.degree. C. until
assayed. Anti-Hin-47 IgG and sIgA titres in samples were determined
by Hin-47-specific ELISA as described above but a goat anti-mouse
IgA antiserum was used in place of the goat anti-mouse IgG
antiserum.
[0187] The serum IgG Hin-47-specific antibody titres following I.G.
immunization is shown in FIG. 9A. These results indicate that an
antigen (non-proteolytic Hin-47 analog) incorporated into PLG,
PLGpZS or PLGpS microparticles was substantially more immunogenic
than soluble antigen of similar dose (4 .mu.g) and better than
antigen that was physically mixed with microparticles when
delivered by the intragastric route. It was experimentally
determined that a dose of soluble antigen (20 .mu.g) which was five
times that which was administered encapsulated within
microparticles (4 .mu.g) was required to elicit an essentially
equivalent response. This result is atypical for most proteins as
there are many examples where in excess of 100 .mu.g of antigen is
required to elicit any significant serum IgG antibody response via
the intragastric route.
[0188] The IgG subtype profile (FIG. 9B) for pooled serum obtained
from the bleeds on day 56 and 78 for microparticle encapsulated
non-proteolytic Hin-47 analog, microparticles physically mixed with
non-proteolytic Hin-47 analog or soluble non-proteolytic Hin-47
analog shows a similar trend as that observed in Example 8. The
IgG1 subtype was dominant when antigen was delivered in soluble
form or when physically mixed with microparticles. Non-proteolytic
Hin-47 analog encapsulated within microparticles exhibits a more
balanced profile with IgG1 and IgG2a more equally represented. Thus
via the intragastric route with antigen encapsulated within
microparticles a more balanced TH.sub.1/TH.sub.2 response was
attainable.
[0189] FIG. 9C shows the results for anti-Hin-47 IgA antibody
responses obtained from bleeds on day 78. With soluble antigen at 4
.mu.g per dose no detectable serum IgA was found, however at 20
.mu.g per dose a few responders were observed. A single significant
response was observed with antigen encapsulated within PLG
microparticles and moderate responses observed for antigen
encapsulated within PLGpZS or PLGpS microparticles. Similarly
modest response was seen for antigen physically mixed with PLG,
PLGpZS or PLGpS microparticles. The highest average levels of serum
IgA were obtained for antigen encapsulated within PLGpS
microparticles.
[0190] The intestinal lavage conducted on day 78 revealed minimal
levels of IgG or sIgA specific for Hin-47 analog in the mucosal
secretions obtained from non-proteolytic Hin-47 analog encapsulated
within PLG, PLGpZS or PLGpS microparticles. This is likely due to
the very low levels of encapsulated antigen administered in this
experiment (4 .mu.g per dose) as oral doses of antigen ranging from
30 .mu.g to 100 .mu.g are usually required to elicit a significant
mucosal response in the absence of any other mucosal adjuvants.
Example 10
[0191] This Example illustrates the immunogenicity of
non-proteolytic Hin-47 analog entrapped in PLG, PLGpZS and PLGpS
microparticles formed in accordance with the present invention, in
mice immunized intranasally.
[0192] Groups of five, 6 to 8 week old female BALB/c mice (Charles
River Breeding Laboratories, Wilmington, Mass.) were immunized
intranasally (I.N.) with the following amounts of antigen in 25
.mu.L of PBS (pH 7.4) on days 1, 7, 14 and 57: PLG, PLGpZS and
PLGpS microparticles prepared as described in Example 5 containing
4.0 .mu.g of non-proteolytic Hin-47 analog; and PLG, PLGpZS and
PLGpS microparticles prepared as described in Example 5 physically
mixed with 4.0 .mu.g non-proteolytic Hin-47 analog (FIG. 10A).
[0193] The mice showed no gross pathologies or behavioral changes
after receiving microparticles that contained encapsulated
non-proteolytic Hin-47 analog or microparticles that were
physically mixed with non-proteolytic Hin-47 analog. Sera were
obtained on days +13, +35, +56 and +78 and were evaluated for the
presence of anti-Hin-47 IgG and IgA antibodies by antigen specific
ELISA as described in Example 8.
[0194] Sera and lung lavage washes were examined for the presence
of Hin-47-specific antibodies. To detect and quantify anti-Hin-47
IgG and sIgA in the respiratory tract, mice were sacrificed by
cervical dislocation, a small incision was made in the trachea and
a tube inserted through the trachea into the lungs where 400 .mu.L
of an enzyme inhibitor solution (0.15 M NaCl, 0.01 M
Na.sub.2HPO.sub.4, 0.005 M EDTA, 0.002 M PMSF, 0.05 U/mL Aprotinin,
and 0.02% v/v NaN.sub.3) was injected. This solution was then
withdrawn, placed on ice and the supernatants clarified by
centrifugation (1000.times.g, 20 minutes) and stored at 0.degree.
C. until assayed. Anti-Hin-47 IgG and sIgA titres in samples were
determined by Hin-47-specific ELISA as described in Example 8.
[0195] The serum IgG Hin-47-specific antibody titres following I.N.
immunization is shown in FIG. 10A. These results indicate that an
antigen (non-proteolytic Hin-47 analog) incorporated into or
physically mixed with PLG, PLGpZS or PLGpS microparticles was more
immunogenic than soluble antigen of similar dose (4 .mu.g).
Essentially equivalent response was obtained for antigen that was
encapsulated or physically mixed with microparticles by the
intranasal route.
[0196] The IgG subtype profile (FIG. 10B) for pooled serum obtained
from the bleeds on day 56 and 78 for microparticle encapsulated
non-proteolytic Hin-47 analog, microparticles physically mixed with
non-proteolytic Hin-47 analog or soluble non-proteolytic Hin-47
analog illustrated a trend different from that shown in Example 8
or Example 9. Non-proteolytic Hin-47 analog encapsulated within
microparticles exhibited a more balanced profile with IgG1, IgG2a
and IgG2b essentially equally represented at either timepoint (day
56 or day 78). For antigen delivered in soluble form or when
physically mixed with microparticles, a similar profile was seen by
day 78 where significant levels of IgG2a can also be seen.
[0197] Thus, via the intranasal route, the TH.sub.1/TH.sub.2
response was essentially the same irrespective of whether antigen
was encapsulated within microparticles, physically mixed with
microparticles or in soluble form.
[0198] FIG. 10C shows the results for anti-Hin-47 IgA antibody
responses obtained from bleeds on day 78. With soluble antigen no
detectable serum IgA was found. However, significant response was
seen for antigen that is encapsulated or physically mixed with
microparticles. The highest levels obtained for antigen either
encapsulated or physically mixed with PLGpS microparticles.
[0199] The lung lavage conducted on day. 78 revealed substantial
differences with respect to IgG and sIgA detected from secretions.
The anti-Hin-47 IgG antibody response (FIG. 10D) from soluble
antigen was negligible, however antigen encapsulated within
microparticles or physically mixed with PLG microparticles elicited
significant levels of IgG from half of each group examined. The
most reproducible response for IgG was found with antigen
physically mixed with microparticles derived from PLGpZS or PLGpS
where most or all mice examined possessed significant levels of
IgG.
[0200] The anti-Hin-47 sIgA antibody response (FIG. 10E) was
similar to that obtained for IgG in FIG. 10D. Soluble antigen
response was negligible with antigen encapsulated within
microparticles or physically mixed with PLG microparticles showing
some response. The most significant response for sIgA was found
with antigen physically mixed with microparticles derived from
PLGpZS or PLGpS where most mice have reasonable levels of sIgA.
[0201] The induction of local protection at mucosal surfaces is
often associated with the presence IgG and sIgA in local
secretions. Whereas it cannot be ruled out that the intranasal
immunization has not also resulted in immunization of the upper
respiratory tract, it is clear that for serum IgG antibody response
encapsulated or physically mixed antigen with microparticles is
effective. To induce local secretions of IgG and sIgA physically
mixing antigen with microparticles and more specifically
microparticles formulated from either PLGpZS or PLGpS appears to be
the more suitable method under these conditions.
Example 11
[0202] This Example illustrates the immunogenicity of Flu-X-31 or
Flu X-31 plus a co-adjuvant BAY R1-005 entrapped in PLG, PLGpZS and
PLGpS microparticles formed in accordance with the present
invention, in mice immunized subcutaneously.
[0203] Groups of six, 6 to 8 week old female BALB/c mice (Charles
River Breeding Laboratories, Wilmington, Mass.) were immunized
subcutaneously (S.C.) with the following amounts of antigen in 250
.mu.L of PBS (pH 7.4) on day 1: PLG, PLGpZS and PLGpS
microparticles prepared as described in Example 5 containing 5.0
.mu.g of Flu X-31 (1.5 .mu.g HA) or 5.0 .mu.g of Flu X-31 (1.5
.mu.g HA) and 50 .mu.g BAY R1-005 when administered in soluble form
or approximately 20 .mu.g BAY R1-005 when co-encapsulated (FIG.
11).
[0204] The mice showed no gross pathologies or behavioral changes
after receiving microparticles that contained encapsulated Flu X-31
or Flu X-31 with BAY R1-005. Sera were obtained on days +21 and +33
and were evaluated for the presence of anti-Flu X-31 IgG antibodies
by antigen specific. ELISA. All samples were analyzed in
duplicate.
[0205] Microtiter plate wells were incubated overnight at room
temperature with 500 .mu.L of 4.0 .mu.g/mL Flu X-31 in 0.05M
carbonate-bicarbonate buffer (pH=9.6). The plates were washed with
PBS+0.05% Tween 20 (operationally defined as washing buffer). Wells
were incubated with 200 .mu.L of 5% skim milk (operationally
defined as blocking buffer). After washing with PBS+0.05% Tween 20,
the plates were incubated for 1 h at 37.degree. C. with 100 .mu.L
of sample serially diluted in blocking buffer. Wells were washed
with PBS+0.05% Tween 20 and 100 .mu.L of HRP-conjugated antibody
(goat anti-mouse IgG (H+L), IgG1, IgG2a or IgG2b) in blocking
buffer was added to each well. After 1 hour incubation at
37.degree. C., the wells were washed 5.times. with PBS+0.05% Tween
20 and 100 .mu.L of freshly prepared colorizing substrate
[H.sub.2O.sub.2 (9 parts) and TMB (1 part)] is added to each well.
After 5 minutes incubation in the dark at room temperature the
reaction is stopped by adding 50 .mu.L of a 2M H.sub.2SO.sub.4
solution and the optical density of the fluid in each well was
determined at 450 nm using a microplate reader. A normal mouse sera
pool was used to establish baseline optical density values in the
assay. Hyperimmune mouse Flu X-31 antiserum was used as a positive
control. Pre-immune sera is used as negative control.
[0206] The serum antibody titres following immunizations are shown
in FIG. 11. The results of a single immunization (day 1) indicated
that antigen presented to the immune system entrapped in PLG,
PLGpZS or PLGpS microparticles was more immunogenic than soluble
antigen alone. The most relevant results were obtained with Flu
X-31 or Flu X-31 plus BAY R1-005 encapsulated within PLGpZS
microparticles. Although a sub-optimal dose of BAY R1-005 was
encapsulated within all microparticles examined, the immunogenic
response with the PLGpZS microparticles also proved to be
significantly higher than that obtained with soluble Flu X-31 and
BAY R1-005 alone.
[0207] The studies presented in this Example demonstrate that viral
antigens from influenza virus can be made more immunogenic and
elicit high levels of serum IgG antibodies, when the antigens are
entrapped in microparticles formed in accordance with the present
invention. In addition the significantly higher immunogenicity
displayed by the microparticle systems after a single immunization
demonstrates the potential of these materials for development as
single dosage vaccines.
Example 12
[0208] This Example illustrates the immunogenicity of Flu-X-31 or
Flu X-31 plus a co-adjuvant BAY R1-005 entrapped in PLG, PLGpZS and
PLGpS microparticles formed in accordance with the present
invention, in mice immunized intranasally.
[0209] Groups of six, 6 to 8 week old female BALB/c mice (Charles
River Breeding Laboratories, Wilmington, Mass.) were immunized
intranasally (I.N.) with the following amounts of antigen in 25
.mu.L of PBS (pH 7.4) on days 1 and 34: PLG, PLGpZS and PLGpS
microparticles prepared as described in Example 5 containing 5.0
.mu.g of Flu X-31 (1.5 .mu.g HA) or 5.0 .mu.g of Flu X-31 (1.5
.mu.g HA) and 50 .mu.g BAY R1-005 when administered in soluble form
or approximately 20 .mu.g BAY R1-005 when co-encapsulated (FIG.
12).
[0210] The mice showed no gross pathologies or behavioral changes
after receiving microparticles that contained encapsulated Flu X-31
or Flu X-31 with BAY R1-005. Sera were obtained on days +21, +33
and +92 and were evaluated for the presence of anti-Flu X-31 IgG
antibodies by antigen specific ELISA as described in Example 10.
All samples were analyzed in duplicate.
[0211] Mice immunized I.N. with soluble antigen, soluble antigen
plus co-adjuvant or encapsulated antigen showed a similar anti-Flu
X-31 IgG antibody response. Interestingly, a reduced (likely due to
delayed release) immunogenic response for encapsulated antigen plus
co-adjuvant relative to soluble antigen, antigen plus adjuvant or
encapsulated antigen on day +21 and +33 was shown when administered
via the nasal route. However, after the second immunization (day
34) a significant boost in response with these groups was observed
resulting in essentially similar immunogenicity on day +92 for all
groups irrespective of adjuvant used.
[0212] The results of the I.N. immunizations described in this
Example shows that the immunogenicity of an antigen or antigen plus
a co-adjuvant cannot be significantly enhanced by entrapment in
microparticles formed in accordance with the present invention.
Example 13
[0213] This Example illustrates the immunogenicity of Flu A-Texas
or Flu A-Texas+BAY R1-005 encapsulated or physically mixed with
microparticles in mice immunized subcutaneously.
[0214] It is known that most non-replicating viral vaccines require
multiple doses for sufficient serum antibody titres to be
protective. Thus, it is strongly desirable to achieve this by
administering a single dose of antigen.
[0215] We sought to examine the immunogenicity of Flu A-Texas or
Flu A-Texas plus a co-adjuvant BAY R1-005 which was entrapped in
PLGpS microparticles or physically mixed with microparticles
administered as a single dose, formed in accordance with the
present invention. Groups of six, 6 to 8 week old female DBA2 mice
(Charles River Breeding Laboratories, Wilmington, Mass.) were
immunized subcutaneously (S.C.) with the following amounts of
antigen in 250 .mu.L of PBS (pH 7.4) on day 1: PLGpS prepared as
described in Example 5 containing 1.0 .mu.g of Flu A-Texas (0.35
.mu.g HA) or 10.0 .mu.g of Flu A-Texas (3.5 .mu.g HA) or 1.0 .mu.g
of Flu A-Texas (0.35 .mu.g HA) and approximately 2.0 .mu.g of BAY
R1-005 or 10.0 .mu.g of Flu A-Texas (3.5 .mu.g HA) and
approximately 20 .mu.g of BAY R1-005 or microparticles prepared as
described in Example 5 physically mixed with 1.0 .mu.g of Flu
A-Texas (0.35 .mu.g HA) and 20 .mu.g of BAY R1-005 or 10.0 .mu.g of
Flu A-Texas (3.5 .mu.g HA) and 20 .mu.g of BAY R1-005 (FIG.
13).
[0216] The core loading of PLGpS microparticles containing Flu
A-Texas and BAY R1-005 was determined via amino acid analysis
(Table 1). The mass of microparticles administered was adjusted
such that the required dose of Flu A-Texas (1.0 .mu.g of Flu
A-Texas (0.35 .mu.g HA) for the low dose groups or 10.0 .mu.g of
Flu A-Texas (3.5 .mu.g HA) for the high dose groups) was delivered.
Thus the dose of BAY R1-005 delivered for the high dose groups was
ten fold greater than that of the low dose groups examined. It is
expected that formulation conditions can be adjusted such that the
quantity of BAY R1-005 co-encapsulated is comparable.
[0217] The mice showed no gross pathologies or behavioral changes
after receiving microparticles that contained encapsulated Flu
A-Texas or Flu A-Texas with BAY R1-005. Sera were obtained on days
+21 and +42 or +57 and were evaluated for the presence of
functional antibody via the hemagglutination inhibition antibody
assay (HAI). All samples were analyzed in duplicate.
[0218] The influenza hemagglutination inhibition antibody assay was
performed with heat-inactivated mouse serum that had been incubated
for 30 minutes with 10% chicken red blood cells to remove
non-specific inhibitors. Twofold dilutions of sera were added to a
96 well microtiter plate and 8 HA units of virus suspension in an
equal volume were added to each well and incubated at room
temperature for 30 minutes. A 1.0% suspension of chicken red blood
cells were added to each well and incubated at room temperature for
30 minutes. Positive and negative reference sera were included in
the test to ascertain specificity and sensitivity of the test.
Positive sera from influenza virus immunized animals. Negative sera
from PBS immunized animals, titre should be less than or equal to
15. The HAI titres are expressed as the reciprocal of the highest
dilution that completely inhibits hemagglutination of
erythrocytes.
[0219] The results of a single immunization (day 1) as shown in
FIG. 13 indicate that by day +42 antigen presented to the immune
system in soluble form or entrapped in PLGpS microparticles (FIG.
13) elicits negligible or low functional antibody. For Flu (10
.mu.g) presented to the immune system as a physical mixture with
BAY R1-005 the HAI response elicited was 8.times. higher than that
obtained with soluble antigen of similar dose and 6.times. higher
that that obtained by PLGpS/Flu microparticles of either dose. The
most relevant results were obtained for PLGpS/Flu(A-Texas)/BAY
R1-005 formulated microparticles wherein the HAI titre of the low
dose group (1 .mu.g) was 8.times. that of soluble antigen and the
high dose group (10 .mu.g) was 32.times. that of soluble antigen. A
moderate increase in the HAI titre was found for the additional
control groups examining physical mixtures of Flu with PLGpS
microparticles or physical mixtures of Flu, PLGpS microparticles
and BAY R1-005. The HAI responses elicited with physically mixed
Flu and PLGpS microparticles was found to be 4.times. higher for
the low dose group and 8.times. higher for the high dose group than
that obtained with soluble antigen of similar dose. The HAI
responses elicited for physical mixtures of Flu with PLGpS
microparticles and BAY R1-005 was found to be 4.times. higher than
that obtained with soluble antigen, irrespective of dose.
[0220] The studies presented in this Example demonstrate that viral
antigens from influenza virus elicit high levels of functional
antibodies when the antigens are entrapped in microparticles in the
presence of an additional adjuvant (as determined by HAI titres),
formed in accordance with the present invention. There is a dose
dependence observed and in addition the significantly higher
functional antibody (a correlate for protection) displayed by the
microparticle antigen and adjuvant co-encapsulated systems after a
single immunization further demonstrate the potential of these
materials for development as single dosage forms.
Example 14
[0221] This Example examines the effects of formulation conditions
typically employed for microencapsulation on individual components
of Flu (trivalent) vaccine.
[0222] Flu (trivalent) vaccine contains three homologous HA strains
(A/Texas, A/Johannesburg and B/Harbin) in approximately equal
amounts. The quantitation of each specific HA component has been
determined by single radial diffusion assay (SRID) (ref. 32). The
SRID assay employs polyclonal sera to detect each HA component and
is an effective indicator for conformational changes. The minimal
concentration detectable with this assay for each component is
approximately 10 .mu.g/mL. A series of samples containing 2.0 mL of
Flu (trivalent) vaccine (concentration=265 .mu.g/mL) were prepared.
For each of these samples, the concentration of A/Texas specific HA
20.25 .mu.g/mL, A/Johannesburg specific HA=20.60 .mu.g/mL and
B/Harbin specific HA=21.42 .mu.g/mL, respectively, was determined
by SRID.
[0223] The effects of solvents typically employed in
microencapsulation procedures, such as Dichloromethane (DCM) or
Ethyl Acetate (EtOAc), were evaluated under conditions that
simulate the homogenization procedure. Short sonication times (30
seconds) were used to model this. Additionally, small amounts of
additives, such as BAY (a glyco-lipo-peptide) and DC-Chol (a
cationic lipid), were examined to determine if improved recovery of
antigen can be confirmed.
[0224] The scale of the reaction was designed to mimic the
procedures as described in Example 5. The organic solvent was
evaporated from each mixture prior to SRID analysis and suitable
controls were examined to determine if the organic solvent,
homogenizing method or addition of BAY or DC-Chol perturbed the
results in any way.
[0225] The results of this study are shown in Table 8. The effects
of sonication on the sample were minimal as shown by comparing
entries 1 and 2. Entries 3 and 4 show that the recovery of antigen
was affected by sonication in organic solvents like EtOAc or DCM.
When EtOAc is employed, 75% of A/Texas specific HA and 78% of
A/Johannesburg specific HA is recovered. No B/Harbin specific HA
component was detectable by this method after treatment with EtOAc.
This result indicates that less than 50% of this component was
actually recovered, as the lower detection limit is about 10
.mu.g/mL. When DCM was employed as solvent, 85% of A/Texas specific
HA, 96% of A/Johannesburg specific HA and less than 50% of B/Harbin
specific HA component were recovered respectively.
[0226] Entries 5 and 6 examine the influence of BAY or DC-Chol as
additive in the organic phase with EtOAc as solvent. For this
combination, all components were detectable (at levels >50%)
with 65% (BAY) or 82% (DC-Chol) A/Texas specific HA recovered, 82%
(BAY) or 70% (DC-Chol) B/Harbin specific HA recovered and 87% (BAY)
or 88% (DC-Chol) A/Johannesburg specific HA recovered respectively.
Entries 7 and 8 examine BAY or DC-Chol as an additive in the
organic phase with DCM as solvent. The assay indicates that some
components were not completely recovered or that the assay is
effected in some way. Specifically, 91% (BAY) or 92% (DC-Chol)
A/Texas specific HA is recovered. Less than 50% B/Harbin specific
HA was recovered in either case. The results for A/Johannesburg
specific HA are inconclusive due to deviations from linearity with
these combinations. This occurred only when DCM was used as
solvent.
[0227] Maximum recovery of all components was obtained for
formulations employing Flu (trivalent) vaccine in EtOAc with BAY or
DC-Chol as additive. This Example indicates that materials with
lipophilic properties, such as BAY or DC-Chol, can be used to
stabilize vaccine formulations containing sensitive components.
Materials with these properties can function at interfaces such
that proteins are protected from the air/water hydrophobic surface
during homogenization. Protein conformation is particularly
sensitive to these effects. The SRID assay results reported in this
Example support these conclusions.
Example 15
[0228] This Example illustrates the single dose immunogenicity of
Flu (trivalent) or Flu (trivalent)+adjuvant cocktails
coencapsulated within PLGpS microparticles in mice immunized
subcutaneously.
[0229] Flu (trivalent) vaccine contains three homologous HA strains
(A/Texas, A/Johannesburg and B/Harbin) in approximately equal
amounts. The total HA content in 10.0 .mu.g of flu (trivalent)
vaccine is 2.35 .mu.g. Each specific HA component has been
determined by single radial diffusion assay (SRID) (ref. 32) to be
0.76 .mu.g A/Texas, 0.81 .mu.g A/Johannesburg and 0.78 .mu.g
B/Harbin respectively.
[0230] In this study the dose of HA administered for each component
strain was significantly less than that used in Example 13. For
comparison, the monovalent vaccine used in Example 13 contained
approximately 3.25 .mu.g of A/Texas specific HA.
[0231] Mice were immunized by the subcutaneous route in the
presence of adjuvants chosen based on Th.sub.2/Th.sub.1 profile;
DC-Chol (a cationic lipid) and BAY R1-005 (glyco-lipo-peptide with
more balanced Th.sub.2/Th.sub.1), PCPP
(poly[di(carboxylatophenoxy)-phosphazene] sodium salt, (Virus
Research Institute, Cambridge Mass.), primarily Th.sub.2. PLGpS
microparticles have been shown to induce a more balanced
Th.sub.2/Th.sub.1 profile, as described in Example 8.
[0232] Groups of eight 6 to 8 week old female DBA-2 mice (Charles
River Breeding Laboratories, Wilmington, Mass.) were immunized
subcutaneously (S.C.) with the following amounts of antigen in 250
.mu.L of PBS (pH 7.4) on day 1: PLGpS microparticles prepared as
described in Example 5 containing 10.0 .mu.g of Flu
(trivalent--2.35 .mu.g HA) or 10.0 .mu.g of Flu (trivalent--2.35
.mu.g HA) and BAY R1-005, DC-Chol or PCPP (Table 5).
[0233] The core loading of PLGpS microparticles containing Flu
(trivalent) was determined via amino acid analysis (Table 6).
Excellent recovery of antigen was found for all PLGpS microparticle
formulations (>70%). This result is consistent with our findings
that the initial concentration of protein solution can dramatically
effect the encapsulation efficiencies.
[0234] The mass of microparticles administered was adjusted such
that the required dose of 10.0 .mu.g of Flu (trivalent--2.35 .mu.g
total HA) was delivered. No attempt to optimize the dose of
adjuvant coadministered was made, although it is expected that this
would be a function of formulation conditions.
[0235] The mice showed no gross pathologies or behavioral changes
after immunization with PLGpS microparticles that contained
encapsulated Flu (trivalent) vaccine or Flu (trivalent) vaccine and
adjuvants cocktails. Sera were obtained on days 21, 42 and 57 and
were evaluated for total IgG, IgG1, IgG2a and for the presence of
functional antibody via the hemagglutination inhibition antibody
assay (HAI). Each strain (A/Texas, A/Johannesburg and B/Harbin) was
examined to evaluate the effects of encapsulation and release on
immunogenicity and functional antibody in a multi-component system.
All samples were analyzed in duplicate. The antibody ELISA's were
performed as described in Example 13.
[0236] FIGS. 14a to c, show the specific IgG antibody responses
elicited by PLGpS/Flu (trivalent) microparticle formulations, to
each strain contained within the Flu (trivalent) vaccine.
[0237] By day 57, the highest total IgG titres were found for
PLGpS/Flu (trivalent)/DC-Chol microparticle formulations. This
result was true for each strain examined (A/Texas--32.times.
response of soluble Flu control, A/Johannesburg--64.times. response
of soluble Flu control, B/Harbin--64.times. response of soluble Flu
control).
[0238] By day 57, slightly higher IgG antibody responses, as
indicated by titre, to A/Texas (2.times. response of soluble Flu
control) and A/Johannesburg (4.times. response of soluble Flu
control) were found for PLGpS/Flu(trivalent)/PCPP microparticle
formulations.
[0239] By day 57, essentially identical total specific IgG antibody
responses (irrespective of strain), as indicated by titre, were
elicited for the PLGpS/Flu (trivalent), PLGpS/Flu (trivalent)/BAY
microparticle formulations and the soluble Flu (trivalent) vaccine
control group. This is consistent with the results obtained in
Example 13 with monovalent vaccine where similar results were
observed for these three systems.
[0240] To determine the influenza hemagglutination inhibition
antibody assay (HAI) for each HA strain, serum samples were heated
at 56.degree. C. for 30 min to inactivate complement and
pre-treated with trypsin (0.1 mL of 8 mg/mL)/periodate (12.5
.mu.L-0.001M) to destroy endogenous inhibitors of hemagglutination.
Serially diluted antisera were tested in duplicate for their
ability to inhibit the agglutination of 1% chick red blood cells by
4 HAU of A/Texas, A/Johannesburg or B/Harbin virus respectively in
a standard HAI assay (ref. 33).
[0241] FIGS. 15a to c, show the specific HAI titres elicited to
each HA strain contained within the trivalent vaccine. By day 57,
the highest sustainable HAI titres were found for PLGpS
microparticles formulated with Flu (trivalent) vaccine in the
presence of DC-Chol (HAI titre; 240 to HA specific A/Texas
(8.times. soluble Flu (trivalent) control), 240 to HA specific
A/Johannesburg (16.times. soluble Flu (trivalent) control) and 60
to HA specific B/Harbin (8.times. soluble Flu (trivalent)
control)). Notably higher titres were also found for PLGpS
micropartilcles formulated with Flu (trivalent) in the presence of
BAY (HAI titre; 60 to HA specific A/Texas (2.times. soluble Flu
(trivalent) control), 60 to HA specific A/Johannesburg (4.times.
soluble Flu (trivalent) control) and 15 to HA specific B/Harbin
(equal to soluble Flu (trivalent) control)). The HAI titres found
for BAY formulations with Flu (trivalent) vaccine, in this Example,
are comparable to those obtained for the low dose groups in Example
13, wherein 1 .mu.g Flu (monovalent) vaccine--0.325 .mu.g A/Texas
specific HA was administered. PLGpS/Flu (trivalent)/PCPP
microparticle formulations failed to elicit any detectable
functional antibody response.
[0242] An experiment was conducted examining a higher dose of
PLGpS/Flu (triv)/DC-Chol formulated microparticles. This group of
mice was immunized on day 1 with approximately twice the dose of
Flu (triv) vaccine administered to the other groups
(Dose-Flu(triv)=21.2 .mu.g, total HA=5.00 .mu.g). The results
obtained for this high dose are included in FIGS. 15a to c. The HAI
titres elicited for a control group immunized with soluble Flu
(triv) vaccine at this dose were marginal and essentially
equivalent to the low dose group examined (results not shown). By
day 57, this formulation elicited significantly higher specific HAI
titres than the low dose group (HAI titre; 480 to HA specific
A/Texas (5.times. soluble Flu (trivalent) control), 480 to HA
specific A/Johannesburg (5.times. soluble Flu (trivalent) control)
and 120 to HA specific B/Harbin (3.times. soluble Flu (trivalent)
control). Additionally, it was found that the HAI titres elicited
were sustainable, being maintained at these levels from day 21
through 57.
[0243] Additives, such as BAY or DC-Chol, were introduced into the
organic phase, containing polymer and organic solvent wherein they
can function at the interface to protect antigen during formulation
procedures (see Example 13, Table 8). This serves to increase the
recovery of antigenic material.
[0244] In addition, in vitro release studies have shown that a
sizable amount of the encapsulated components are released within
the first three days. It has been experimentally determined that
about 5 to 15% of antigen encapsulated is detectable at this stage
of release (antigen which is surface localized on the
microparticle). The priming immunization was significantly enhanced
by the co-release of adjuvants. This is likely the result of being
localized in close proximity to antigen during the initial release
phase.
[0245] It is noteworthy that PLGpS microparticle formulations
coencapsulating antigens with adjuvants, such as PCPP, did not
result in formulations with improved vaccine efficacy. This
material was added to the aqueous phase of the primary emulsion and
does not possess the lipophilic properties of DC-Chol or BAY. It
has been demonstrated that PCPP has strong adjuvant properties in
other systems, yet in this Example, a marginal improvement in
encapsulation efficiency or adjuvancy of the microparticle
formulation is observed.
[0246] The studies presented in this Example demonstrate that viral
antigens from influenza virus in a multi-component system can
elicit high levels of total IgG antibody and functional antibodies
(HAI) when the Flu antigens are entrapped in microparticles in the
presence of an additional co-encapsulated adjuvant, such as BAY or
DC-Chol.
Example 16
[0247] This Example evaluates the infection rate and protection
after single dose subcutaneous immunization with PLGpS
microparticle formulations in the mouse model.
[0248] A/Taiwan/1/86 (H1N1) as live influenza viruses in
egg-derived allantoic fluid, mouse-adapted A/Taiwan/l/86 and
commercial A/Taiwan/l/86 monovalent subunit vaccine (Fluzone.RTM.)
were obtained from Pasteur Mrieux, Connaught, USA (Swiftwater,
Pa.).
[0249] Groups of 8 female DBA-2 mice aged 6 to 8 weeks (described
in Example 14) were immunized subcutaneously on day 1 with Flu
(trivalent) vaccine containing A/Texas, A/Johannesburg and B/Harbin
strains (2.35 .mu.g total HA in 0.25 mL PBS). Control mice received
either PBS alone or 400 hemagglutination units (HAU) of live
A/Texas virus as allantoic fluid. Fourteen days later, mice were
challenged intranasally while under anesthesia with 50 .mu.L of
live mouse-adapted A/Taiwan/1/86 (5 LD50) in allantoic fluid.
Protection was assessed by monitoring mortality daily and morbidity
(weight change) every 2 days up to 14 days post-challenge.
[0250] FIG. 16 shows the results of the challenge. As expected, all
mice immunized with homologous live (A/Texas) virus suffered
minimal loss in weight and fully recovered within 2 weeks of
challenge as measured by the weight percent change from baseline.
Also as expected, mice immunized with PBS suffered a precipitous
drop in weight which by day 10 fell below 30% of baseline. No mice
in this control group survived after 10 days.
[0251] Mice immunized with PLGpS/Flu (trivalent)/DC-Chol
microparticle formulations were successfully infected, as indicated
by the 8% average drop in weight within 4 days post challenge. Of
the PLGpS formulated groups examined, these mice exhibited the
swiftest recovery, reaching 100% of baseline after 2 weeks. The
entire group recovered indicating a protective titre was raised in
each mouse.
[0252] Mice immunized with PLGpS/Flu (trivalent)/BAY microparticle
formulations were also successfully infected, as indicated by the
10% average drop in weight by day 10. These mice recovered more
slowly, reaching 95% of baseline after 2 weeks. The entire group
recovered indicating that a protective titre was raised in all of
these mice, although the rate of recovery indicates the level of
protection was less than that seen for the DC-Chol group at this
dose. This is in accordance with the HAI values determined in
Example 15 for these two groups.
[0253] The soluble Flu (trivalent) vaccine control group and the
PLGpS/Flu (trivalent) or PLGpS/Flu (trivalent)/PCPP microparticle
formulations were less successful. These groups experienced a 23 to
29% average drop in weight by days 8-10 post challenge. The rate of
recovery was slowest for these mice reaching 86 to 92% of baseline
after 2 weeks. Additionally not all mice in these groups survived.
Six of eight mice survived from the PLGpS/Flu (trivalent)
microparticle formulated group. Seven of eight survived from the
PLGpS/Flu (trivalent)/PCPP formulated group and six of eight
survived from the soluble Flu (trivalent) control group. In each of
these cases no or low protection is observed.
[0254] In Example 14, we evaluated the effects of
microencapsulation formulation conditions on the Flu (trivalent)
vaccine. Strain-specific (A/Texas, A/Johannesburg and B/Harbin)
analysis of antigen recovery by SRID suggested that minimal loss in
antigenic activity for all three strains in this multi-component
system could be expected when employing DC-Chol or BAY in the
organic phase with PLGpS polymer and EtOAc as solvent.
[0255] In Example 15, the higher functional antibody (a correlate
for protection) elicited by single subcutaneous administration of
PLGpS/Flu (trivalent) vaccine formulated in the presence of DC-Chol
or BAY demonstrated the utility of these formulations in the mouse
model. The results of the challenge/protection study, described in
Example 16, are in agreement with the ranking for protection as
suggested by the functional antibody studies.
[0256] The delivery of antigen and adjuvant within a biodegradable
microparticle can result in more efficient presentation to the
immune system. The adjuvancy found for soluble mixtures of DC-Chol
or BAY with antigen(s) may be significantly increased by employing
this formulation strategy. The possibility for fewer and lower
(antigen/adjuvant) dose regimens is indicated by these results.
Specifically, this Example strongly indicates the potential of
these new microparticle based formulations for development as a
single efficacious dosage form.
Example 17
[0257] This Example illustrates the immunogenicity of transferrin
binding protein (Tbp-2) derived from Moraxella catarrhalis (as
described in WO 97/13785, assigned to the assignee hereof and the
disclosure of which is incorporated herein by reference)
encapsulated in PLGpS microparticles, Tbp-2 physically mixed with
PLGpS microparticles or Tbp-2 formulated with Alum in mice
immunized subcutaneously.
[0258] Groups of five, 6 to 8 week old female BALB/c mice (Charles
River Breeding Laboratories, Wilmington, Mass.) were immunized
subcutaneously (S.C.) with the following amounts of antigen in 250
.mu.L of PBS (pH 7.4) on days 1, 28 and 43: PLGpS microparticles
prepared as described in Example 5 containing 0.3 .mu.g of Tbp-2;
PLGpS microparticles prepared as described in Example 5 physically
mixed with 0.3 .mu.g of Tbp-2; and Alum (1.5 mg/dose) formulated
with 0.3 .mu.g of Tbp-2 (Table 5).
[0259] The mice showed no gross pathologies or behavioral changes
after receiving microparticles that contained encapsulated Tbp-2,
microparticles physically mixed with Tbp-2 or Alum physically mixed
with Tbp-2. Sera were collected on days +14, +27, +42 and +55 and
were evaluated for the presence of anti-Tbp-2 IgG antibodies by
antigen specific ELISA as described in Example 8. All samples were
analysed in duplicate.
[0260] The core loading of PLGpS microparticles containing tbp-2
was determined via amino acid analysis (4.0 .mu.g/mg (32.8%)) as
described in Example 6.
[0261] The results of immunizations (FIG. 17a) indicate that
antigen presented to the immune system entrapped in PLGpS
microparticles elicited a substantially higher titre than that
obtained for soluble antigen or for antigen physically mixed with
PLGpS microparticles alone. In addition, this study indicates that
microencapsulated formulations were as immunogenic as the
traditional Alum adjuvanted systems. The kinetics of the immune
response were found to be similar for both formulations.
[0262] The IgG subtype profile (FIG. 17b) for the bleed obtained on
day 55 revealed differences in the immune responses elicited for
Tbp-2 encapsulated in PLGpS microparticles and Tbp-2 physically
mixed with PLGpS microparticles or formulated with Alum. IgG1 is
the dominant subtype detected (with some IgG2b) when antigen was
administered in soluble form or as a physical mixture with PLGpS
microparticles or formulated with Alum.
[0263] In this Example, the IgG subtypes induced by Tbp-2
encapsulated within PLGpS microparticles elicit comparatively more
IgG2a. This trend is a similar trend to that observed for Hin-47 in
Example 8.
[0264] These results suggest that the mechanisms of immune response
induced by immunizing with encapsulated antigens are different from
that generated with Alum. A more balanced Th.sub.2/Th.sub.1 profile
(as indicated by the IgG2a:IgG1 ratio) is exhibited when antigen is
administered encapsulated within microparticles.
[0265] As may be seen from the results herein, the quality of the
immune response mediated by PLGpS microparticles encapsulating
antigen is substantially different from that obtained by physically
mixing with PLGpS microparticles, formulating with Alum or by
administering soluble antigen alone. Additionally, the magnitude of
the immune response induced by Alum in antigen/Alum formulations is
comparable to that provided by microparticles containing
encapsulated antigen.
Example 18
[0266] This Example illustrates the immunogenicity of Tbp-2
encapsulated in PLGpS microparticles and Tbp-2 physically mixed
with PLGpS microparticles in mice immunized intranasally.
[0267] Groups of five, 6 to 8 week old female BALB/c mice (Charles
River Breeding Laboratories, Wilmington, Mass.) were immunized
intranasally (I.N.) with the following amounts of antigen in either
10 .mu.L or 50 .mu.L of PBS (pH 7.4) on days 1, 28 and 43: PLGpS
microparticles prepared as described in Example 5 containing 6.0
.mu.g of Tbp-2; and PLGpS microparticles prepared as described in
Example 5 physically mixed with 6.0 .mu.g Tbp-2 (Table 5).
[0268] The mice showed no gross pathologies or behavioral changes
after immunization with PLGpS microparticles that contained
encapsulated Tbp-2 or PLGpS microparticles that were physically
mixed with Tbp-2. Sera were obtained on days 14, 27, 42 and 55 and
were evaluated for the presence of anti-Tbp-2 IgG antibodies by
antigen specific ELISA as described in Example 8. All samples were
analyzed in duplicate.
[0269] The core loading of PLGpS microparticles containing Tbp-2
was determined via amino acid analysis (4.0 .mu.g/mg (32.8%)) as
described in Example 6.
[0270] The serum IgG Tbp-2-specific antibody titres following I.N.
immunization is shown in FIG. 18. When the volume of the dose
administered was low (10 .mu.L) these results indicate that an
antigen (Tbp-2) incorporated into or physically mixed with PLGpS
microparticles is less immunogenic than soluble antigen of similar
dose (6.0 .mu.g).
[0271] When the volume of the dose was increased (50 .mu.L), the
overall titres are significantly higher for all groups. These
results indicate that an antigen (Tbp-2) physically mixed with
PLGpS microparticles is substantially more immunogenic than
microparticle encapsulated antigen or for soluble antigen of
similar dose (6.0 .mu.g) in accordance with earlier observations
made for intranasal immunization of Hin-47 (Example 10).
[0272] In our initial study with intranasal Hin-47 immunizations,
Example 10, it was found that a strong humoral response and robust
secretory response were obtained by administering Hin-47 physically
mixed with microparticles. This study with Tbp-2 has confirmed the
earlier observations. Additionally, we have established that the
volume of the dose administered plays a significant role in the
type and strength of the immune response invoked.
Example 19
[0273] This Example illusrates the release of antigen from the
microparticles.
[0274] Having established that antigen encapsulated within PLGpS
microparticles is as immunogenic as Alum formulations, we next
sought to examine whether the prime and delayed release character
of antigens encapsulated within these polymeric matrices could be
exploited so that fewer immunizations would be needed.
[0275] In this Example, we examined the immunogenicity of Tbp-2
encapsulated in PLGpS microparticles, physically mixed with PLGpS
microparticles or formulated with Alum or CFA/IFA in guinea pigs
immunized subcutaneously.
[0276] Groups of two, 6 to 8 week old female guinea pigs (Charles
River Breeding Laboratories, Wilmington, Mass.) were immunized
subcutaneously (S.C.) with the following amounts of antigen: PLGpS
microparticles encapsulating 5.0 .mu.g of tbp-2 suspended in 500
.mu.L of PBS (pH 7.4) on days 1 and 28, prepared as described in
Example 5; Complete Freunds Adjuvant (CFA) formulated in PBS (pH
7.4) with 5.0 .mu.g of Tbp-2 on day 1 followed intramuscularly
(I.M.) by Incomplete Freunds Adjuvant (IFA) formulated in PBS (pH
7.4) with 5.0 .mu.g of Tbp-2 on days 14 and 28; or Alum formulated
in PBS (pH 7.4) with 5.0 .mu.g of Tbp-2 on days 1, 14 and 28 (Table
5).
[0277] The guinea pigs showed no gross pathologies or behavioral
changes after immunization with PLGpS microparticles that contained
encapsulated Tbp-2, PLGpS microparticles physically mixed with
Tbp-2, CFA/IFA formulated Tbp-2 or Alum formulated Tbp-2. Sera were
obtained on days 40 and 56 and were evaluated for the presence of
anti-Tbp-2 IgG antibodies by antigen specific ELISA. All samples
were analyzed in duplicate. The antibody ELISA's are described in
Example 8.
[0278] The core loading of PLGpS microparticles containing Tbp-2
was determined via amino acid analysis (4.0 .mu.g/mg (32.8%)) as
described in Example 6.
[0279] The results shown in FIG. 19 indicate that two doses of
PLGpS microencapsulated Tbp-2 elicited similar anti-Tbp-2 antibody
responses as 3 doses of Tbp-2 formulated either in CFA/IFA or with
Alum.
[0280] The present results strongly suggest that microencapsulated
Tbp-2 may be exploited as a candidate vaccine for two dose
schedules.
Example 20
[0281] This Example illustrates the immunogenicity of rUrease or
rUrease/adjuvant cocktails encapsulated within PLGpS microparticles
in mice immunized subcutaneously or intragastrically.
[0282] Groups of eight, 6 to 8 week old outbred Swiss female mice
(Janvier, France) were immunized subcutaneously (S.C.) with the
following amounts of antigen in 300 .mu.L of PBS (pH 7.4) on days 1
and 28 and 56: PLGpS microparticles prepared as described in
Example 5 containing 10.0 .mu.g of rUrease; and PLGpS
microparticles prepared as described in Example 5 coencapsulating
10.0 .mu.g of rUrease and DC-Chol, PCPP or CT-X; 10.0 .mu.g of
rUrease plus soluble DC-Chol (65 .mu.g/dose) or PCPP (100
.mu.g/dose) as controls or intragastrically (I.G.) with the
following amounts of antigen in 300 .mu.L of 0.15 M NaHCO.sub.3 (pH
9.0) on days 1, 28 and 56: PLGpS microparticles prepared as
described in Example 5 containing 40.0 .mu.g of rUrease; and PLGpS
microparticles prepared as described in Example 5 coencapsulating
40.0 .mu.g of rUrease and DC-Chol, PCPP or CT-X (Table 5).
[0283] The core loading of PLGpS microparticles containing rUrease
was determined via amino acid analysis or polyclonal rUrease
specific ELISA (Table 7) as described in Example 6. The polyclonal
ELISA assay performed on PLGpS microparticle extracts typically
provides a measure of total protein (recovered epitope)
encapsulated. Control experiments have allowed us to quantify the
extent to which the solvent/base (SDS) extraction procedure effects
the integrity of the antigen. Under the conditions employed we have
estimated that about 65 to 75% of the extracted protein remains
fully detectable by this assay. Thus this determination will be
lower than the measure of total protein recovered via amino acid
analysis (AAA). Suitable controls were included to ensure that the
presence of the adjuvants (DC-Chol, CT-X or PCPP) did not interfere
with the results obtained. The presence of DC-Chol did not seem to
influence the assay, however, PCPP provided anomalous values which
were uncharacteristically higher when compared to the total protein
by AAA in some analyses. In the case of CT-X, a separate polyclonal
ELISA assay was developed to quantify the amount of CT-X
coencapsulated.
[0284] The mass of microparticles administered was adjusted such
that the required dose of 10.0 .mu.g of rUrease (subcutaneously) or
40.0 .mu.g of rUrease (orally) was delivered.
[0285] The mice showed no gross pathologies or behavioral changes
after immunization with PLGpS microparticles that contained either
encapsulated rUrease or coencapsulated mixtures of rUrease and
adjuvant. Sera were obtained on day 85 and were evaluated for the
presence of anti-rUrease IgG1 and IgG2a antibodies by antigen
specific ELISA. All samples were analyzed in duplicate.
[0286] ELISA's were performed according to standard protocols
(biotinylated conjugates, streptavidine peroxidase complex were
from Amersham and o-phenylenediamine dihydrochloride (OPD)
substrate from Sigma). Plates were coated overnight at 4.degree. C.
with H. pylori extracts (5 .mu.g/mL) in 0.05M carbonate-bicarbonate
buffer (pH 9.6). After saturation with BSA (Sigma), plates were
incubated with sera (1.5 hrs), biotinylated conjugate (1.5 hrs),
strepavidin peroxidase complex (1 h) and substrate (OPD--10 min). A
polyclonal mouse serum directed against H. pylori extract served as
a control in each experiment. The titres were expressed as the
inverse of the dilution giving 50% of the maximal absorbance value
at 492 nm. Pre-immune sera is used as negative control.
[0287] Mice were immunized by the subcutaneous or intragastric
route in the presence of adjuvants chosen based on
Th.sub.2/Th.sub.1 profile (DC-Chol--more balanced
Th.sub.2/Th.sub.1, PCPP--primarily Th.sub.2) or for known mucosal
adjuvancy (Cholera Toxin (CT-X) or heat labile enterotoxin from E.
coli (LT)). PLGpS microparticles have been shown to induce a more
balanced Th.sub.2/Th.sub.1 profile relevant to other typical
adjuvants, such as Alum.
[0288] The IgG subtype profile for pooled bleeds obtained on day 85
after subcutaneous immunizations is shown in FIG. 20. In all cases,
the IgG1 response was higher than the IgG2a response (IgG2a:IgG1
ratio ranges from 0.08 to 0.33) when PLGpS/rUrease microparticle
formulations were administered subcutaneously. The total IgG1
response for the soluble control groups (rUrease+DC-Chol or PCPP)
was in the same absolute range. There are differences noted for the
IgG2a responses between adjuvant systems that were either
coencapsulated with antigen or administered as a physical mixture
with antigen.
[0289] For PLGpS/rUrease microparticles (IgG2a:IgG1=0.33),
PLGpS/rUrease/CT-X (IgG2a:IgG1=0.30) and PLGpS/rUrease/DC-Chol
(IgG2a:IgG1=0.20), a more balanced IgG2a:IgG1 antibody response
(indicative of Th.sub.2/Th.sub.1 ratio) was obtained. For
comparison, the rUrease+DC-Chol control group IgG2a:IgG1=0.03.
[0290] For PLGpS/rUrease/PCPP microparticle formulations, primarily
a Th.sub.2 response is noted IgG2a:IgG1 ratio=0.08. The analogous
soluble mixture of rUrease+PCPP is strongly Th.sub.2 biased with a
very low IgG2a:IgG1 ratio=0.003.
[0291] In general, coencapsulation of antigen in the presence of
these adjuvants tends to shift the typical immunological profile
towards a more balanced Th.sub.2/Th.sub.1 response.
[0292] Immunization via the intragastric route in most cases did
not elicit any detectable systemic response. Notable exceptions to
this are the positive control with LT having a modest systemic
response (IgG2a:IgG1 ratio=0.1) and for rUrease/PLGpS
microparticles coencapsulating CT-X (IgG2a:IgG1 ratio=2.1).
Interestingly, for oral immunizations, encapsulated antigen plus
mucosal adjuvant induces a strikingly different systemic antibody
response. The ratio of IgG2a:IgG1 is now strongly in favor of IgG2a
indicative of the Th.sub.1 path. Examination of the literature
reveals that CT-X co-administered with antigen orally typically
induces an immune response similar to that elicited by LT.
Primarily an IgG1 or TH.sub.2 type of response is reported (ref.
34). This study illustrates that the quality of the immune response
may be changed as a consequence of coencapsulation in PLGpS
microparticles.
Example 21
[0293] This Example evaluates the infection rate and protection
after subcutaneous or oral immunizations with PLGpS
microparticle/rUrease formulations in the mouse model.
[0294] Mice were challenged 6 weeks after the 2'nd boost (Day 85)
by gastric gavage with 300 .mu.L of a suspension of H. pylori
bacteria (3.times.10.sup.6 cfu). Based on in vitro release studies
(Example 7), antigen release from microparticles requires
approximately 4 weeks, thus the challenge was scheduled for 2 weeks
after this time point.
[0295] Four weeks after the challenge, mice were killed and
stomachs were sampled to evaluate urease activity (Jatrox test,
Procter and Gamble) in a sterile flow hood. Urease activity was
assessed 24 hours post-mortem by measuring the absorbance at 550
nm. The principle of the test is that the urea present in the test
medium is split by Helicobacter pylori urease. The rise in pH
causes a color change in the indicator which is likewise present in
the test medium (phenol red), from yellow to pink red.
[0296] Mice were infected with a streptomycin-resistant
mouse-adapted strain (X43-2AN) of H. pylori. The infection rate was
reproducible; 100% of the mice were infected in all experiments, as
judged by urease activity measured in the stomach (Jatrox test). It
is to be noted that the streptomycin resistance of the challenge
strain allows it to grow on a highly selective medium, making this
test very sensitive (no contaminants coming from the normal
flora).
[0297] This strain was stored at -70.degree. C. in Brucella Broth
(BB) (Biomrieux) supplemented with 20% v/v glycerol and 10% v/v
foetal bovine serum (FBS)-(Hyclone). The challenge suspension was
prepared as follows: for pre-culture, H. pylori was grown on
Mueller-Hinton Agar (MHA; Difco) containing 5% v/v sheep blood
(Biomerieux) and antibiotics: Thrimethoprim (5 .mu.g/mL),
Vancomycin (10 .mu.g/mL), Polymixin B sulphate (5 .mu.g/mL),
Amphotericin (5 .mu.g/mL) and Streptomycin (50 .mu.g/mL) selective
marker of H. pylori strain X43-2AN (TVPAS) All antibiotics were
purchased from Sigma. MHA-TVPAS plates were incubated for 3 days at
37.degree. C. under micro-aerobic conditions. The pre-culture was
used to innoculate a 75 cm.sup.2 vented flask (Costar) containing
50 mL of BB supplemented with 5% V/v FBS and all antibiotics
(TVPAS). The flask was kept under micro-aerobic conditions with
gentle shaking for 24 hrs. The suspension was characterized by
Gram's staining, urease activity (Urea indole medium, Diagnostic
Pasteur), catalase (H.sub.2O.sub.2, 3% v/v) and oxidase activity
(Biomerieux discs). Viability and motility were checked by phase
contrast microscopy. The suspension was diluted in BB to O.D. 550
nm=0.1 (which was equivalent to 1.times.10.sup.7 CFU/mL).
[0298] In this study, mice for which an OD. <0.1 was obtained
were considered protected or at least have less than 10.sup.3 to
10.sup.4 bacteria/stomach (compared to 10.sup.5 to 10.sup.6 in
controls). Groups of mice representing unimmunized/infected and
unimmunized/uninfected controls were also included in the study
(results not shown).
[0299] FIG. 21a, shows that protection (via the subcutaneous
route), as judged by the assay described above, follows the ranking
PLGpS/rUrease/DC-Chol (geometric mean 0.092)>PLGpS/rUrease/CT-X
(geometric mean=0.105), PLGpS/rUrease (geometric mean 0.163) and
PLGpS/rUrease/PCPP (geometric mean 0.269). The LT+rUrease (oral)
positive control group (geometric mean=0.136) is the standard for
protection in this study. Interestingly, typical results with
rUrease plus soluble DC-Chol (geometric mean=0.600) or rUrease plus
PCPP (geometric mean=1.07) control groups clearly show the
advantages of coencapsulating antigen and adjuvant as a PLGpS
microparticle formulation.
[0300] Mann-Whitney statistical analysis of the data presents the
following conclusions. The PLGpS/rUrease/DC-Chol and
PLGpS/rUrease/CT-X formulations were not significantly different
from the LT+rUrease positive control group. The PLGpS/rUrease/PCPP,
rUrease+DC-Chol and rUrease+PCPP groups were significantly
different from these groups (p<0.01) and the PLGpS/rUrease group
was significantly different from the PLGpS/rUrease/PCPP,
rUrease+DC-Chol and the rUrease+PCPP (p<0.01) groups.
[0301] From this analysis, the PLGpS/rUrease/DC-Chol (7/8 mice have
OD. <0.1) and PLGpS/Urease/CT-X (5/8 mice have OD. <0.1)
microparticle groups exhibited solid protection whereas the
PLGpS/rUrease (2/8 mice have OD. <0.1) microparticle group and
the rUrease+DC-Chol group (2/10 OD. <0.1) exhibited moderate
protection and the PLGpS/rUrease/PCPP (0/8 mice have OD. <0.1)
microparticle group and the rUrease+PCPP groups (0/10 OD. <0.1)
exhibited low or no protection respectively. This ranking tends to
follow the more balanced Th.sub.2/Th.sub.1 ratios as determined
from immunogenicity studies (Example 20).
[0302] FIG. 21b shows that no full protection via the oral route
was observed for all groups of mice immunized by PLGpS
microparticles containing additional adjuvants.
[0303] Statistical ranking follows the order PLGpS/rUrease/CT-X
(geometric mean=0.229)>PLGpS/rUrease/DC-Chol (geometric mean
0.403), PLGpS/rUrease (geometric mean=0.475) and PLGpS/rUrease/PCPP
(geometric mean=0.493). The LT+rUrease (oral) group (geometirc mean
0.136) is the positive control for this study.
[0304] Mann-Whitney statistical analysis of the data presents the
following conclusions. The LT+rUrease positive control group was
significantly different than the PLGpS/rUrease/DC-Chol and
PLGpS/rUrease/CT-X formulations (p<0.01). The
PLGpS/rUrease/DC-Chol and PLGpS/rUrease/CT-X formulations were not
significantly different from each other. The PLGpS/rUrease and the
PLGpS/rUrease/PCPP formulations were not significantly different
form each other.
[0305] In this study, the PLGpS/rUrease/CT-X (2/8 mice have OD.
<0.1) and the PLGpS/rUrease/DC-Chol (1/8 mice have OD. <0.1)
microparticle formulations exhibited partial protection.
Comparatively, the positive control group of rUrease plus LT
exhibited solid protection (7/8 mice have OD. <0.1).
[0306] It was suggested, in Examples 14 to 16, that the
presentation of adjuvant to the immune system by association with
delivery vehicles, such as PLGpS, microparticles increases the
adjuvants effectiveness resulting in a more efficacious
vaccine.
[0307] Notably, typical control studies in this Example have
examined immunogenicity and protection after immunizations with
soluble mixtures of adjuvants, such as DC-Chol or PCPP, and rUrease
by the subcutaneous route, and have found that protection was low
to moderate. These results provide additional evidence that the
presentation of antigen and adjuvant in the form of a particulated
matrix can result in substantially more efficacious vaccines.
[0308] Additional benefits, such as reduced adjuvant toxicity and
the possibility of modulating the quality of the immune response
which is characteristic for the adjuvant employed, have been
demonstrated in this Example. It was experimentally determined that
the IgG subtype antibody responses to rUrease obtained after oral
immunization with PLGpS/rUrease/CT-X formulated microparticles was
uncharacteristically in favor of IgG2a (Th.sub.1 path).
[0309] In the case of coencapsulated antigen and CT-X (a known
mucosal adjuvant) administered subcutaneously or orally, it is also
likely that this material stabilizes the antigen within the
polymeric matrix during formulation, storage and release by a
mechanism similar to that observed for microparticles prepared in
the presence of HSA or BSA (ref. 35).
[0310] In conclusion, this study demonstrates the feasibility and
efficacy of PLGpS microparticle based systemic immunization to
induce significant protection against H. pylori infection in the
mouse model. This study also demonstrates the feasibility for PLGpS
microparticle based oral immunization to induce partial protection
against H. pylori infection in the mouse model.
[0311] Additionally, co-encapsulation of rUrease in the presence of
additional adjuvants (specifically DC-Chol or CT-X), can result in
notable improvement in vaccine efficacy.
SUMMARY OF THE DISCLOSURE
[0312] In summary of this disclosure, the present invention
provides a particulate carrier for an agent, particularly one
having biological activity, comprising a matrix of polymer and
biologically active material. The particulate carriers in the form
of microparticles are able to efficiently deliver agents to the
cells of the immune system of a subject following mucosal or
parenteral administration to produce an immune response.
Modifications are possible within the scope of this invention.
1 TABLE 1 Antigen(s) entrapped in PLG, PLGpZS or PLGpS
microparticles Antigen Preparation Hin-47 (1.55 mg/mL) + BAY 800
.mu.L of Hin-47 in aqueous (5.0 mg/mL) internal phase plus 40.0 mg
of BAY in the organic phase rD-15 (2.05 mg/mL) 800 .mu.L of rD-15.
Hin-47 (1.95 mg/mL) + rD-15 400 .mu.L of Hin-47 plus 400 .mu.L
(1.95 mg/mL) of rD-15. Flu X-31 (2.0 mg/mL) 800 .mu.L of Flu X-31
or 800 .mu.L Flu A-Texas (1.48 mg/mL) of Flu A-Texas Flu (2.0
mg/mL) + BAY 800 .mu.L of Flu X-31 or 800 .mu.L (5.0 mg/mL) of Flu
A-Texas in aqueous Flu A-Texas (1.48 mg/mL) + BAY internal phase
plus 40.0 mg of (5.0 mg/mL) BAY in the organic phase
[0313]
2TABLE 2 Summary of Microparticle Core Loading and Encapsulation
Efficiencies (Hin-47, Hin-47 + Bay R1-005) Epitope Equiv. Protein
HIN-47 Total Protein Epitope Total Adjuvant (Conc.) via ELISA.sup.1
via AAA.sup.2 vs Total via AAA.sup.2 (Adjuvants) Polymer (Encaps.
Eff.).sup.3 (Encaps. Eff.).sup.3 Protein % (Encaps. Eff.).sup.3
Hin-47 PLG 1.4 ug/mg 2.8 ug/mg 50% (1.55 mg/mL) (10.2%) (20.4%)
PLGpZS 5.3 ug/mg 7.5 ug/mg 71% 30.8% (43.5%) PLGpS 1.6 ug/mg 3.3
ug/mg 48% (11.6%) (23.9%) Hin-47 PLG 2.5 ug/mg 3.8 ug/mg 66% 13.5
(3.4%) (1.55 mg/mL) (20.1%) (30.6%) Bay-R1005 PLGpZS 4.1 ug/mg 5.5
ug/mg 75% 15.6 (3.9%) (5.0 mg/mL) (19.8%) (26.6%) PLGpS 6.1 ug/mg
9.2 ug/mg 66% 23.2 (4.6%) (39.4%) (59.4%) .sup.1ELISA values are
averages of 2 independent measurements of protein obtained after
complete hydrolysis of microparticles. .sup.2AAA values are
averages of 2 independent measurements on protein (or adjuvant)
encapsulated within microparticles and 2 independent measurements
on protein (or adjuvant) obtained after complete hydrolysis of
microparticles. .sup.3Encapsulation efficiency (Encaps. Eff.)
calculated as follows: 1 total mass of protein ( or adjuvant )
recovered total amount of protein ( or adjuvant ) used .times. 100
%
[0314]
3TABLE 3 Summary of Microparticle Core Loadings and Core Loading
and Encapsulation Efficiencies (rD-15, rD-15 + Hin-47) Epitope
Equivalent Total Protein Protein Hin-47 via ELISA.sup.1 via
AAA.sup.2 (conc.) Polymer (Encaps. Eff.).sup.3 (Encaps. Eff.).sup.3
rD-15 PLG 7.8 ug/mg (43.7% (1.95 mg/mL) PLGpZS 7.1 ug/mg (39.2%)
PLGpS 7.3 ug/mg (43.7%) rD-15 PLG 1.6 ug/mg (8.0%) 7.0 ug/mg
(35.1%) (1.95 mg/mL) + PLGpZS 5.5 ug/mg (26.1%) 13.1 ug/mg (62.1%)
Hin-47 PLGpS 2.4 ug/mg (10.8%) (11.6 ug/mg (52.1%) (1.95 mg/mL)
.sup.1ELISA values are averages of 2 independent measurements of
protein obtained after complete hydrolysis of microparticles.
.sup.2AAA values are averages of 2 independent measurements on
protein encapsulated within microparticles and 2 independent
measurements on protein obtained after complete hydrolysis of
microparticles. .sup.3Encapsulation efficiency (Encaps. Eff.)
calculated as follows: 2 total mass of protein recovered total
amount of protein used .times. 100 %
[0315]
4TABLE 4 Summary of Microparticle Core Loadings and Encapsulation
Efficiencies for Flu X-31 + Bay R1-005 and Flu A-Texas, Flu A-Texas
+ Bay R1-OO5 Protein Total Protein Total Adjuvant (Conc.) via
AAA.sup.1 via AAA.sup.1 (Adjuvants) Polymer (Encaps. Eff.).sup.2
(Encaps. Eff.).sup.2 Flu X-31 PLG 4.7 ug/mg (26.1%) (2.0 mg/mL)
PLGpZS 6.1 ug/mg (34.1%) PLGpS 7.1 ug/mg (39.7%) Flu X-31 PLG 5.6
ug/mg (35.1%) 22.9 ug/mg (5.2%) (2.0 mg/mL PLGpZS 8.4 ug/mg (52.5%)
28.1 ug/mg (10.0%) Bay R1005 PLGpS 5.0 ug/mg (31.0%) 27.4 ug/mg
(8.4%) (5.0 mg/mL) Flu-A-Texas PLGPS 2.7 ug/mg (22.8%) (1.48 mg/mL)
Flu A-Texas PLGpS 3.4 ug/mg (31.6%) 11.5 ug/mg (3.5%) (1.48 mg/mL)
Bay-R1005 (5.0 mg/mL) .sup.1AAA values are averages of protein (or
adjuvant) isolated after complete hydrolysis of microparticles and
protein (or adjuvant) encapsulated within microparticles.
.sup.2Encapsulation efficiency (Encaps. Eff.) calculated as
follows: 3 total mass of protein ( or adjuvant ) recovered total
amount of protein ( or adjuvant ) used .times. 100 %
[0316]
5TABLE 5 Summary of Microparticle Formulation Procedures
Antigen(s)/Adjuvants entrapped in PLGpS microparticles Antigen
Preparation tbp-2 (2.88 mg/ml) 800 .mu.l tbp-2 (diluted to 1.44
mg/ml) in aqueous phase with PBS Flu (Trivalent) (0.533 mg/ml) 800
.mu.L Flu (trivalent) in aqueous internal phase Flu (Trivalent)
(0.533 mg/ml) + 800 .mu.L Flu (trivalent) in aqueous BAY (3.3
mg/ml) internal phase 40.0 mg of BAY in the organic phase Flu
(Trivalent) (0.533 mg/ml) + 800 .mu.L Flu (trivalent) in aqueous
DC-Chol (3.3 mg/ml) internal phase 40.0 mg of DC-Chol in the
organic phase Flu (Trivalent) (0.533 mg/ml) + 800 .mu.L of combined
solution in aqueous PCPP (1.25 mg/ml) internal phase rUrease (1.00
mg/ml) 800 .mu.L rUrease in aqueous internal phase rUrease (1.00
mg/ml) + 800 .mu.L of combined solution in aqueous CT-X (0.5 mg/ml)
internal phase rUrease (1.00 mg/ml) + 800 .mu.L rUrease in aqueous
internal DC-Chol (3.3 mg/ml) phase 40.0 mg of DC-Chol in the
organic phase rUrease (1.00 mg/ml) + 800 .mu.L of combined solution
in aqueous PCPP (1.25 mg/ml) internal phase
[0317]
6TABLE 6 Summary of Microparticle Core Loadings and Encapsulation
Efficiencies for Flu (trivalent) and Flu (trivalent) + Adjuvants
Protein Total Protein (Conc.) via AAA.sup.1 (Adjuvants) Polymer
(Encapsulation Efficiency).sup.2 Flu (trivalent) PLGpS 5.0 ug/mg
(93.3%) (0.533 mg/mL) Flu (trivalent) PLGpS 5.8 ug/mg (104.6%)
(0.533 mg/mL) Bay-R1005 (3.3 mg/mL) Flu (trivalent) PLGpS 3.9 ug/mg
(72.7%) (0.533 mg/mL) DC-Chol (3.3 mg/mL) Flu (trivalent) PLGpS 4.6
ug/mg (86.6%) (0.533 mg/mL) PcPp (1.25 mg/mL) .sup.1AAA values are
averages of protein isolated after complete hydrolysis of
microparticles and protein encapsulated within microparticles.
.sup.2Encapsulation efficiency calculated as follows: 4 total mass
of protein recovered total amount of protein used .times. 100 %
[0318]
7TABLE 7 Summary of Microparticle Core Loadings and Encapsulation
Efficiencies for rUrease and rUrease + Adjuvants Protein Total
Protein Total Protein (Conc.) via AAA.sup.1 via ELISA.sup.2
(Adjuvants) Polymer (Encaps. Eff.).sup.3 (Encaps. Eff.).sup.3
rUrease PLGpS 3.44 ug/mg (45.8 %) 2.5 ug/mg (33.2%) (1.0 mg/mL)
rUrease PLGpS 3.15 ug/mg (44.0%) 2.2 ug/mg (30.3%) (1.0 mg/mL)
DC-Chol (3.3 mg/mL) rUrease PLGpS 7.35 ug/mg (N/D %).sup.4 rUrease
= (1.0 mg/mL) 4.5 ug/mg (53.0%) CT-X CT-X = 2.5 ug/mg (0.5 mg/mL)
(59.0%) rUrease PLGpS 5.97 ug/mg (67.2%) 3.3 ug/mg (37.1%).sup.5
(1.0 mg/mL) PcPP (1.25 mg/mL) .sup.1AAA values are averages of two
independent measurements of protein encapsulated within
microparticles, as described in Example 6. .sup.2polyclonal ELISA
(capture sandwich antigen assay) conducted on microparticle
hydrosylates, as described in Example 6. Average of 4
determinations. .sup.3Encapsulation efficiency (Encaps. Eff.)
calculated as follows: 5 total mass of protein recovered total
amount of protein used .times. 100 % .sup.4N/D = not determinable
by AAA as total protein is actually a combination of rUrease and
CT-X. .sup.5ELISA determination in the presence of PCPP was highly
variable.
[0319]
8TABLE 8 Flu (trivalent) Formulations Examined by SRID's Flu SRID
Samples Solvent/Solution Sonicate Additives
A/Texas:B/Harbin:A/Johannes conc. = 265 ug/mL examined (30 sec)
(quantity) (ug/mL) Entry #1 none no -- 20.25:20.6:21.42 Entry #2
none yes -- 21.73:19.8:23.05 Entry #3 EtOAc yes -- 15.21:N/D:16.73
Entry #4 DCM yes -- 17.28:N/D:20.54 Entry #5 EtOAc yes BAY
13.04:16.8:18.63 (20 .mu.g) Entry #6 EtOAc yes DC-Chol
16.54:14.4:18.79 (20 .mu.g) Entry #7 DCM yes BAY 18.44:N/D:N/L (20
.mu.g) Entry #8 DCM yes DC-Chol 18.55:N/D:N/L (20 .mu.g) N/D = not
determined (below detection limit of .about.10 .mu.g/mL). N/L =
test failed or was not reproducible under experimental
conditions.
REFERENCES
[0320] 1. Levine, M. M.; Cryz, S.; Sorenson, K.; Kaper, J.;
Wasserman, S. S.; Burr, D.; Lim, Y. L.; Clemens, J.; Rifai, A. R.;
Totosudirgo, H.; Losonsky, G.; Heppner, D. G.; Punjabi, N.; Witham,
N.; and Simanjuntak, C., Lancet, 340, 1992, 689.
[0321] 2. Eldridge, J. H.; Hammond, C. J.; Meulbroek, J. A.; Staas,
J. K.; and Gilley, R. M., J. Control. Release, 11, 1990, 205.
[0322] 3. Eldridge, J. H.; Tice, T. R.; Meulbroek, J. A.; Staas, J.
K.; McGhee, J. R.; and Gilley, R. M., Mol. Immunol., 28, 1991,
287.
[0323] 4. Hagan, D. T.; Palin, K. J.; and Davis, S. S., Vaccine, 7,
1989, 213.
[0324] 5. Mitsunobu, O.; Synthesis, 1981.
[0325] 6. Zhou, Q., and Kohn, J., J. Macromolecules, 23, 1990,
3300.
[0326] 7. Brode, G. L., Koleske, J. V., J. Macromol. Sci-Chem., A6,
1972, 1109.
[0327] 8. U.S. Pat. No. 2,676,945, Higgins, H. A., Condensation
Polymers of Hydroxyacetic Acid, (1954).
[0328] 9. U.S. Pat. No. 3,839,297, Wasserman, D. and Versfeit, C.
C., Use of Stannous Octoate Catalyst in the Manufacture of
L-(-)-Lactide-Glycolide Copolymer Sutures, (1974).
[0329] 10. Kohn, F. E.; Ommen, J. G., and Feigen, J., Eur. Polym.
J., 19, 1983, 1081.
[0330] 11. Kohn, F. E.; Van Den Berg, J. W. A.; and Van De Ridder,
G., Journal of Applied Polymer Science, 29, 1984, 4265.
[0331] 12. Kricheldorf, H. R.; and Dunsing, R., Polymer Bulletin,
14, 1985, 491.
[0332] 13. Kricheldorf, H. R.; Jonte, J. M.; and Berl, M.,
Macromol. Chem. Suppl., 12, 1985, 25.
[0333] 14. Leenslag, J. W.; and Pennings, A. J., Makromol. Chem.,
188, 1987, 1809.
[0334] 15. Kricheldorf, H. R.; and Sumbel, M., Eur. Polymer J., 25,
1989, 585.
[0335] 16. Hayashi, T.; and Iwatsuki, M., Biopolymers, 29, 1990,
549.
[0336] 17. Hayashi, T.; Likuza, Y.; Oya, M.; and Iwatsuki, M., J.
Appl. Polym. Sci., 43, 1991, 2223.
[0337] 18. Hayashi, T.; likuza, Y.; Oya, M.; and Iwatsuki, M.,
Polym. J., 5, 1993, 481.
[0338] 19. Kohn, J.; and Langer, R., J. Am. Chem. Soc., 109, 1987,
817.
[0339] 20. Yonezawa, N.; Toda, F.; Hasegawa, M., Makromol. Chem.
Rapid Commun., 6, 1985, 607.
[0340] 21. Helder, J.; and Feijen, J., Makromol. Chem. Rapid
Commun., 7, 1986, 193.
[0341] 22. Veld, P. J. A.; Dijkstra, P. J.; Lochem, J. H. van; and
Feigen, J.,. Makromol. Chem., 191, 1990, 1813.
[0342] 23. Langer, R.; Barrera, D. A.; Zylstra, E.; and Lansbury,
P. T., J. Am. Chem. Soc., 115, 1993, 11010.
[0343] 24. Barrera, D. A.; Zylstra, E.; and Lansbury, P. T.,
Macromolecules., 28, 1995, 425.
[0344] 25. P.C.T. Int. Appl. 94 09760, Barrera, D.; Langer, R. S.;
Lansbury, P. T. Jr.; and Vacanti, J. P., Biodegradable Polymers for
Cell Transportation, (1994).
[0345] 26. Veld, P. J. A.; Dijkstra, P. J.; Zheng-Rong, S.;
Gijsbert, T. A. J.; and Feigen, J., J. Polymer Sci., 32(6), 1994,
1063.
[0346] 27. Reed, A. M. and Gilding, D. K.; Polymer, 22, 1981,
494.
[0347] 28. Greene, T. W.; and Wuts, P., Protective Groups in
Organic Synthesis II, 335-338, John Wiley and Sons, Inc., New York,
1991.
[0348] 29. U.S. Pat. No. 4,855,283 granted to Lockhoff et. al.,
1989.
[0349] 30. Wiesmuller, Vaccine, 8, 1989, 29.
[0350] 31. Huang, L. and Gao, X., Biochemical and Biophysical
Research Communications, 179, 1991, 280
[0351] 32. Wood, J. M. et. al.; Development of Biological Standard,
1977, 39, 193-200.
[0352] 33. Palmer, D. F., Coleman, M. T., Dowdle, W. R. and Schild,
G. C.; "Advanced laboratory techniques for influenza diagnosis",
immunology series no. 6, US Dept. Health, Education and Welfare.
Washington D.C.; 1975, 51-52.
[0353] 34. Ruedl, C., Rieser, C., Kofler, N., Wick, G. and Wolf,
H.; Vaccine, 1996, 14, 792-798.
[0354] 35. Lu, W. and Park, T. G.; Journal of Pharmaceutical
Technology, 1995, 49, 13-19.
[0355] 36. Gopferich, A.; Biomaterials; 17, 1996, 103.
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