U.S. patent application number 10/761571 was filed with the patent office on 2005-07-21 for novel phytol derived immunoadjuvants and their use in vaccine formulations.
Invention is credited to Ghosh, Swapan K..
Application Number | 20050158329 10/761571 |
Document ID | / |
Family ID | 34750195 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158329 |
Kind Code |
A1 |
Ghosh, Swapan K. |
July 21, 2005 |
Novel phytol derived immunoadjuvants and their use in vaccine
formulations
Abstract
This invention relates to a novel immunoadjuvant, an adjuvant
component, and vaccines containing the adjuvant component. The
adjuvant includes phytol or a phytol derivative. The adjuvant
component, when combined with a soluble or particulate antigen,
provides a vaccine with an enhanced ability to induce both humoral
and cytotoxic immune responses while displaying reduced toxicity
and/or adverse side effects over vaccines that include the antigen
but without the benefit of this adjuvant component.
Inventors: |
Ghosh, Swapan K.; (Terre
Haute, IN) |
Correspondence
Address: |
WOODARD, EMHARDT, MORIARTY, MCNETT & HENRY LLP
BANK ONE CENTER/TOWER
111 MONUMENT CIRCLE, SUITE 3700
INDIANAPOLIS
IN
46204-5137
US
|
Family ID: |
34750195 |
Appl. No.: |
10/761571 |
Filed: |
January 21, 2004 |
Current U.S.
Class: |
424/184.1 ;
514/724 |
Current CPC
Class: |
A61K 31/045 20130101;
A61K 39/39 20130101; A61K 2039/55511 20130101 |
Class at
Publication: |
424/184.1 ;
514/724 |
International
Class: |
A61K 039/00; A61K
031/045 |
Claims
1. A composition comprising: a vaccine preparation effective for
treatment of a mammal in unit dosage form including: an effective
amount of an antigen; an adjuvant component comprising phytol,
isophytol, or a phytol derivative, said antigen homogenously
dispersed in the adjuvant component; and optionally a carrier.
2. The composition of claim 1 wherein the adjuvant component
comprises phytol.
3. The composition of claim 1 wherein the adjuvant component
comprises isophytol.
4. The composition of claim 1 wherein the adjuvant component
comprises phytanol.
5. The composition of claim 1 wherein the adjuvant component
comprises a phytol derivative selected from the group consisting
of: phytanol; 1-chloro-3,7,11,15-tetramethyl hexadec-2-ene;
iodo-3,7,11,15-tetramethyl hexadeca-2-ene;
1-amino-3,7,11,15-tetramethyl hexadec-2-ene;
3,7,11,15-tetramethyl-hexadec-2-en-1-al;
3,7,11,15-tetramethyl-1-hexadeca- nal;
3,7,11,15-tetramethyl-1-hexadec-2-enyl acetate;
3,7,11,15-tetramethyl-1-hexadecanyl acetate;
1-chloro-3,7,11,15-tetrameth- yl hexadecane;
1-iodo-3,7,11,15-tetramethyl hexadecane;
1-amino-3,7,11,15-tetramethyl hexadecane; 1-methyl
amino-3,7,11,15-tetramethyl hexadecane; stereoisomers and mixtures
thereof.
6. The composition of claim 1 wherein the adjuvant component
comprises phytol or a phytol derivative of the formula I or II:
5wherein R.sup.1 is selected from the group of chemical moieties,
ions, or radicals consisting of: Br.sup.-, Cl.sup.-; I.sup.-;
--NH.sub.2, --NO.sub.2, OH, PO.sub.4.sup.32, HPO.sub.4.sup.-,
NHR.sup.2, OC(O)R.sup.2, OR.sup.2, wherein R.sup.2 is a hydrocarbyl
having 1 to 10 carbon atoms or a carbohydrate.
7. The composition of claim 1 wherein the antigen includes a
T-independent antigen.
8. The composition of claim 7 wherein the antigen is selected from
the group consisting of: polysaccharides, pneumococcus
polysaccharides, bacterial lipopolysaccharides, synthetic
lipoolysacchrarides, and hapten-polysaccharide conjugates.
9. The composition of claim 1 wherein the antigen includes a
T-dependent antigen.
10. The composition of claim 9 wherein the antigen is selected from
the group consisting of: proteins, peptides, lipoproteins,
glycoproteins, gangliosides, cerebrosides, nucleoproteins,
eukaroytic cellular isolates, and prokaryotic cellular
isolates.
11. The composition of claim 1 wherein the carrier is sterile water
at pH 7.0.
12. The composition of claim 1 wherein the carrier is comprises
physiological buffers that include carbonates, bicarbonates,
phosphates.
13. The composition of claim 1 wherein the vaccine composition is
an oil-in-water emulsion.
14. The composition of claim 13 comprising a surfactant or
emulsifier.
15. The composition of claim 14 wherein the emulsifier is selected
from the group consisting of: phospholipids, lysophosphoglycerides,
spingomyelin, phosphatidyl choline, phosphatidyl ethanolamine,
phosphatidyl inositol and mixtures thereof.
16. The composition of claim 1 wherein the vaccine composition
comprises the phytol or the phytol derivative and the antigen in a
weight ratio of between about 1:4 to about 1:1.
17-26. (canceled)
27. A composition comprising a vaccine preparation in unit dosage
form including an effective amount of an antigen conjugated
directly to phytanol or a phytol derivative and a surfactant mixed
in equal volume, and optionally a carrier or buffer solution.
28. The composition of claim 27 comprising between 4 and 100
micrograms of the antigen conjugated directly to phytanol or a
phytol derivative.
29. The composition of claim 27 comprising between about 0.05 to
about 0.1% (wt/v) of the surfactant.
30. A composition comprising: a vaccine preparation in unit dosage
form including: an effective amount of an antigen; an adjuvant
component comprising a phytol derivative; and optionally a liquid
carrier.
31. The composition of claim 30 wherein the adjuvant component
comprises a phytol derivative selected from the group consisting
of: phytanol; 1-chloro-3,7,11,15-tetramethyl hexadec-2-ene;
iodo-3,7,11,15-tetramethyl hexadeca-2-ene;
1-amino-3,7,11,15-tetramethyl hexadec-2-ene;
3,7,11,15-tetramethyl-hexadec-2-en-1-al;
3,7,11,15-tetramethyl-1-hexadeca- nal;
3,7,11,15-tetramethyl-1-hexadec-2-enyl acetate;
3,7,11,15-tetramethyl-1-hexadecanyl acetate;
1-chloro-3,7,11,15-tetrameth- yl hexadecane;
1-iodo-3,7,11,15-tetramethyl hexadecane;
1-amino-3,7,11,15-tetramethyl hexadecane; 1-methyl
amino-3,7,11,15-tetramethyl hexadecane; stereoisomers and mixtures
thereof.
32. The composition of claim 30 wherein the adjuvant component
comprises a phytol derivative of the formula I: 6wherein R.sup.1 is
selected from the group of chemical moieties, ions, or radicals
consisting of: Br.sup.-, Cl.sup.-; I.sup.-; --NH.sub.2, --NO.sub.2,
OH, PO.sub.4.sup.32, HPO.sub.4.sup.-, NHR.sup.2, OC(O)R.sup.2, and
OR.sup.2, wherein R.sup.2 is a hydrocarbyl having 1 to 10 carbon
atoms or a carbohydrate.
33. The composition of claim 32 wherein R.sup.1 is selected from
--NH.sub.2 or NHR.sup.2 wherein R.sup.2 is a hydrocarbyl having 1
to 10 carbon atoms or a carbohydrate.
34. The composition of claim 32 wherein R.sup.1 is selected from
the group of chemical moieties consisting of: OH, OC(O)R.sup.2, and
OR.sup.2, wherein R.sup.2 is a hydrocarbyl having 1 to 10 carbon
atoms or a carbohydrate.
35. The composition of claim 32 wherein R.sup.1 is selected from
the group of chemical moieties consisting of: OH, PO.sub.4.sup.32,
and HPO.sub.4.sup.-.
36. The composition of claim 32 wherein R.sup.1 is selected from
the group of chemical moieties consisting of: Br.sup.-, Cl.sup.-;
and I.sup.-.
37. The composition of claim 32 comprising an emulsifier selected
from the group consisting of: phospholipids such as
phosphoglycerides, lysophosphoglycerides, spingomyelin,
phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl
inositol and mixtures thereof.
38. The composition of claim 32 comprising a surfactant selected
from the group consisting of: mannide monooleates, 1,4-sorbitan,
mono- and triesters, and polyoxyethylene derivatives of stearic
acid.
39. The composition of claim 32 wherein the antigen is selected
from the group consisting of: polysaccharides, pneumococcus
polysaccharides, bacterial lipopolysaccharides, synthetic
lipoolysacchrarides, and hapten-polysaccharide conjugates.
40. The composition of claim 32 wherein the antigen is selected
from the group consisting of: proteins, peptides, lipoproteins,
glycoproteins, ganliosides, cerebrosides, nucleoproteins,
eukaroytic cellular isolates, and prokaryotic cellular
isolates.
41. The composition of claim 30 wherein the adjuvant component
comprises a phytol derivative of the formula II: 7wherein R.sup.1
is selected from the group of chemical moieties, ions, or radicals
consisting of: Br.sup.-, Cl.sup.-; I.sup.-; --NH.sub.2, --NO.sub.2,
OH, PO.sub.4.sup.32, HPO.sub.4.sup.-, NHR.sup.2, OC(O)R.sup.2,
OR.sup.2, wherein R.sup.2 is a hydrocarbyl having 1 to 10 carbon
atoms or a carbohydrate.
42. The composition of claim 41 wherein R.sup.1 is selected from
--NH.sub.2 or NHR.sup.2 wherein R.sup.2 is a hydrocarbyl having 1
to 10 carbon atoms or a carbohydrate.
43. The composition of claim 41 wherein R.sup.1 is selected from
the group of chemical moieties consisting of: OH, OC(O)R.sup.2, and
OR.sup.2, wherein R.sup.2 is a hydrocarbyl having 1 to 10 carbon
atoms or a carbohydrate.
44. The composition of claim 41 wherein R.sup.1 is selected from
the group of chemical moieties consisting of: OH, PO.sub.4.sup.32,
and HPO.sub.4.sup.-.
45. The composition of claim 41 wherein R.sup.1 is selected from
the group of chemical moieties consisting of: Br.sup.-, Cl.sup.-;
and I.sup.-.
46. The composition of claim 41 comprising an emulsifier selected
from the group consisting of: phosphoglycerides,
lysophosphoglycerides, spingomyelin, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl inositol and mixtures
thereof.
47. The composition of claim 41 comprising a surfactant selected
from the group consisting of: mannide monooleates, 1,4-sorbitan,
mono- and triesters, and polyoxyethylene derivatives of stearic
acid.
48. The composition of claim 41 wherein the antigen is selected
from the group consisting of: polysaccharides, pneumococcus
polysaccharides, bacterial lipopolysaccharides, synthetic
lipoolysacchrarides, and hapten-polysaccharide conjugates.
49. The composition of claim 41 wherein the antigen is selected
from the group consisting of: proteins, peptides, lipoproteins,
glycoproteins, ganliosides, cerebrosides, nucleoproteins,
eukaroytic cellular isolates, and prokaryotic cellular
isolates.
50. A method of enhancing the immunogenicity of a vaccine
composition, said method comprising: selecting an antigen eliciting
a desired immunogenic response in a mammal, and combining the
antigen with the phytol derivative of claim 32 in a physiological
acceptable carrier.
51. A method of treating a patient, said method comprising:
preparing a vaccine formulation containing an antigen eliciting a
desired immunogenic response in a mammal, and combining the antigen
with the phytol derivative of claim 32 in a physiological
acceptable carrier; and administering the vaccine formulation to
the patient.
52. A method of enhancing the immunogenicity of a vaccine
composition, said method comprising: selecting an antigen eliciting
a desired immunogenic response in a mammal, and combining the
antigen with the phytol derivative of claim 41 in a physiological
acceptable carrier.
53. A method of treating a patient, said method comprising:
preparing a vaccine formulation containing an antigen eliciting a
desired immunogenic response in a mammal, and combining the antigen
with the phytol derivative of claim 41 in a physiological
acceptable carrier; and administering the vaccine formulation to
the patient.
54. The composition of claim 1 wherein the adjuvant component
comprises phytol or a phytol derivative of the formula I or II:
8wherein R.sup.1 is selected from the group of ions consisting of:
Br.sup.-, Cl.sup.-, and I.sup.-.
55. The composition of claim 1 wherein the adjuvant component
comprises phytol or a phytol derivative of the formula I or II:
9wherein R.sup.1 is selected from the group of chemical moieties
consisting of: --NH.sub.2, --NO.sub.2, and NHR.sup.2, wherein R is
a hydrocarbyl having 1 to 10 carbon atoms or a carbohydrate
56. The composition of claim 1 wherein the adjuvant component
comprises phytol or a phytol derivative of the formula I or II:
10wherein R.sup.1 is selected from the group of chemical moieties,
ions, or radicals consisting of: OH, OC(O)R.sup.2, OR.sup.2,
wherein R.sup.2 is a hydrocarbyl having 1 to 10 carbon atoms or a
carbohydrate.
Description
BACKGROUND
[0001] The present invention relates to immunoadjuvants that
include phytol or a phytol derivative and the use of the
immunoadjuvants in a pharmaceutical formulation to effect treatment
by augmenting immunogenicity and effectiveness of vaccines.
[0002] Vaccines are used to provide effective immunity to prevent
infection and even cancer. With the introduction of widespread
immunization in the last century, diseases such as diphtheria,
tetanus, polio, small pox, and pertussis have been significantly
reduced if not eliminated. Moreover, new generations of vaccines
are constantly being sought after for treatment of diseases such as
AIDS, hepatitis, and herpes.
[0003] Vaccines are meant to stimulate the individual's specific
and non-specific immune responses. Vaccines are accompanied by an
adjuvant which can increase the efficacy of the vaccine by a
variety of actions, including retaining the vaccine in the body or
at the site of injection for a significantly long period of time,
eliminating or reducing degradation and/or excretion, and
recruiting or directing macrophages and other antigen-presenting
cells (APCs) to augment humoral and/or cellular immune responses to
the pathogen or associated antigens. However, many adjuvants, while
effective, are also often toxic to humans. These adjuvants can
cause severe adverse inflammatory reactions. Therefore, while
adjuvants can be effective in enhancing the immunogenicity of the
antigens in the vaccines, they can also induce severe side
reactions.
[0004] The only adjuvant currently accepted by the FDA for use in
humans is aluminum hydroxide or alum. While this adjuvant presents
fewer instances of adverse reactions, it is not as effective in
inducing humoral or cellular immune responses as other adjuvants,
such as incomplete Freund's adjuvant (IFA) or complete Freund's
adjuvant (CFA, which is IFA plus mycobacterial components). Both
Freund's adjuvants, for example, are used in animals (other than
humans) to produce antibodies for treatment and diagnosis under
strict regulations of USDA and Public Health Service (PHS). While
CFA/IFA are not approved for use in humans because they can be
quite toxic to certain individuals, they are still considered as
the gold standard in adjuvant formulation. In countries other than
the United States, IFA is used in many vaccine formulations.
[0005] A safer, non-toxic, non-carcinogenic adjuvant which elicits
an enhanced immune response for the antigens in the vaccine
formulation would be of immense value to both human and animal
welfare. The present invention addresses the above described
problems and provides a variety of benefits and advantages.
SUMMARY OF THE INVENTION
[0006] The present invention relates to immunoadjuvants that
include phytol, isophytol, or a phytol derivative and the use of
the immunoadjuvants to elicit an immune response. Various aspects
of the invention are novel, nonobvious, and provide various
advantages. While the actual nature of the invention covered herein
can only be determined with reference to the claims appended
hereto, certain forms and features, which are characteristic of the
preferred embodiments disclosed herein, are described briefly as
follows.
[0007] In one form, the present invention provides a composition
that includes a vaccine preparation in unit dosage form comprising:
an effective amount of an antigen; an adjuvant component comprising
phytol, isophytol, or a phytol derivative; and optionally a
carrier.
[0008] In another form, the present invention provides a method of
enhancing the immunogenicity of a vaccine composition. The method
comprises selecting an antigen that induces a desired immunogenic
response in a mammal; combining the antigen with phytol, isophytol,
or a phytol derivative in a physiologically acceptable carrier; and
treating a mammal with the vaccine composition in an amount
effective to induce the immunogenic response. In preferred
embodiments, the vaccine formulation includes the antigen in a
lower effective dose than another vaccine formulation that includes
the same antigen but without the benefit of or in the absence of
the phytol or a phytol derivative. The preferred vaccine
formulation still elicits the desired immunogenic response
substantially the same as the vaccine that does not include the
phytol, isophytol, or phytol derivative.
[0009] In another form, the present invention provides a method for
inducing full spectra of immune responses, namely antibody, NKT
cells, and helper-cytotoxic effector T lymphocytes to soluble and
insoluble antigens. The method comprises selecting an antigen that
induces a desired immunogenic response in a mammal; combining the
antigen with phytol, isophytol, or a phytol derivative in a
physiologically acceptable carrier; and treating a mammal with the
vaccine composition in an amount effective to induce the
immunogenic response.
[0010] In still yet another form, the present invention provides a
method of treating a patient in need thereof with a vaccine. The
method comprises administering to the patient a vaccine composition
comprising an antigen and an adjuvant component including phytol,
isophytol, or a phytol derivative in a physiologically acceptable
carrier.
[0011] Further objects, features, aspects, forms, advantages and
benefits shall become apparent from the description and drawings
contained herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a scheme illustrating various synthetic strategies
for synthesizing phytol derivatives.
[0013] FIG. 2 is a scheme illustrating various synthetic strategies
for synthesizing reduced phytol or phytanol (H-phytol)
derivatives.
[0014] FIGS. 3A-3F are scanned images of spleens obtained from
BALB/c mice treated with adjuvants: A, (PBS); B, pristane; C,
Incomplete Freund's adjuvant (IFA); D, phytol; E, Complete Freund's
adjuvant (CFA); and F, reduced phytol (phytanol).
[0015] FIG. 4 is a time line illustrating the treatment regime used
to evaluate the peritoneal plasmoacytomagenesis in BALB/c mice
treated with pristane, phytol, or phytanol.
[0016] FIGS. 4A-4C are scanned images of mice injected with the
following: A, pristane; B, phytol; and C, phytanol; indicating that
plasmacytoma was not observed in the mice treated with phytol or
phytanol.
[0017] FIG. 5 is a graph illustrating the side effects due to
autoimmunity (anti-DNA antibody response) elicited from various
adjuvants.
[0018] FIG. 6 is a graph comparing various commercially available
adjuvants with phytol derivatives for an anti-phthalate antibody
response.
[0019] FIG. 7 is a graph illustrating the comparison of the various
adjuvants including phytol and phytanol to induce IgG2a antibody
iostype response.
[0020] FIG. 8 is a graph illustrating the induction of
tumor-specific killer T cells by both phytol and phytanol.
DETAILED DESCRIPTION OF THE INVENTION
[0021] For the purposes of promoting an understanding of the
principles of the invention, reference will now be made to the
embodiments illustrated herein and specific language will be used
to describe the same. It will nevertheless be understood that no
limitation of the scope of the invention is thereby intended. Any
alterations and further modifications in the described
formulations, compositions, and/or treatments, and any further
applications of the principles of the invention as described
herein, are contemplated as would normally occur to one skilled in
the art to which the invention relates.
[0022] The present invention provides a novel immunoadjuvant or an
immunoadjuvant component for use in a pharmaceutical or vaccine
formulation. The adjuvant component can include phytol or a phytol
derivative. The vaccine formulation can include, in addition to the
adjuvant or adjuvant component, one or more soluble or membranous
antigens, and optionally one or more of a carrier unlinked, or
covalently or hydrophobically linked, an emulsifier, and a surface
active agent. The vaccine formulation and immunoadjuvant prepared
according to the present invention can be used to treat mammals
including humans either prophylatically or therapeutically as
desired or or deemed medically expedient.
[0023] The adjuvant component for use in the present invention can
include phytol, isophytol, phytanol, and/or a phytol derivative.
Phytol is a commercially available compound that is derived from
vitamin E (a-tocopherol) or from chlorophyll as well as other
naturally occurring species. Phytol can be considered a fatty
alcohol or, more specifically, a branched chain fatty alcohol and
includes an unsaturated beta hydroxy group. (See FIGS. 1A and 1B
for phytol and isophytol, respectively.) 1
[0024] Phytol derivatives as described herein include reduced
phytol (or H-phytol/phytanol) and reduced isophytol in which the
unsaturated group in each species has been reduced with hydrogen.
Reduced phytol or H-phytol will hereafter be referred to as
phytanol, unless otherwise stated. The double bond in phytol (or
isophytol) can be reduced using palladized carbon and hydrogen gas
under standard conditions. FIG. 1 provides a synthetic scheme for
the preparation of representative examples of various phytol
derivatives that are included within the scope of the present
invention. FIG. 2 provides a similar scheme for the preparation of
various examples of reduced phytol derivatives that are included
within the scope of the present invention. Similar synthetic
strategies are envisioned to prepare isophytol derivatives.
Examples of the phytol derivatives include halogenated phytol,
halogenated phytanol, an aminated phytol or phytanol, and phytol or
phytanol derivatives in which the hydroxy group has been
substituted with a nitro group, aldehyde group, or esterified
phytol or phytanol, in which hydroxyl group has been used to
esterify a carboxylic acid to form an ester. Other phytol
derivatives can be carried out using standard procedures of organic
chemistry. It will be understood that either phytol, isophytol or
reduced phytol, reduced isophytol can be used in the following
examples. Examples of the various transformations include oxidation
of the hydroxyl group using pyridine chlorochromate (PCC) or
activated MnO.sub.2. Amine phytol derivatives can be prepared with
phytol aldehydes (from PCC reaction) using a suitable primary or
secondary amine and Raney Ni catalyst. The amine derivatives can be
used to prepare antigen-phytol conjugates; for example, phytolated
lysine or polylysine can be used directly or as a conjugate with
different antigens with lysine serving as a linker. Halogenated
phytol derivatives can be prepared by substituting the hydroxyl
group with chlorine using PCl.sub.3 in DMF (or PCl.sub.5) or
bromine using PBr.sub.3 in pyridine or with iodine using iodine or
sodium iodide and a Lewis base, such as, boron trifluoride etherate
(BF.sub.3--OEt.sub.2), triphenyl phosphine, or phosphorous metal.
The halogenated phytol derivatives also can be used to react with
carbohydrates to prepare a phytol-carbohydrate (or
phytanol-carbohydrate) complex. Examples of preferred carbohydrates
that can be coupled with phytol (or phytanol) include:
monoschharides, oligosachharides, polysachharides, mannitol,
sorbitol, glycosaminoglycans, chondrosine, and arabans. Various
phytol derivatives can be prepared according to established
procedures, for example, suitable synthetic conditions for
preparing the phytol derivatives can be ascertained from Richard
Larock, "Comprehensive Organic Transformations", VCH Publishers,
Inc., 1989, and references cited therein.
[0025] Representative examples of phytol derivatives for use in the
present invention include: hydrogenated phytol
(3,7,11,15-tetramethyl-1-h- exadecanol); chlorinated phytol
(1-chloro-3,7,11,15-tetramethyl hexadec-2-ene); iodinated phytol
(iodo-3,7,11,15-tetramethyl hexadeca-2-ene); aminated phytol
(1-amino-3,7,11,15-tetramethyl hexadec-2-ene);
3,7,11,15-tetramethyl-hexadec-2-en-1-al;
3,7,11,15-tetramethyl-1-hexadecanal;
3,7,11,15-tetramethyl-1-hexadec-2-en- yl acetate;
3,7,11,15-tetramethyl-1-hexadecanyl acetate;
1-chloro-3,7,11,15-tetramethyl hexadecane;
1-iodo-3,7,11,15-tetramethyl hexadecane;
1-amino-3,7,11,15-tetramethyl hexadecane; 1-methyl
amino-3,7,11,15-tetramethyl hexadecane and stereoisomers, and
mixtures thereof.
[0026] The immunoadjuvant component can contain phytol or the
phytol derivative in amounts from between about 100 wt % and about
20 wt %. Other species in the immunoadjuvant component can include
other adjuvants such as aluminum hydroxide, IFA, vegetable oil, and
the like.
[0027] The adjuvant component can be combined with a wide variety
of antigens to provide the vaccine formulation. For example, the
vaccine formulation can include antigens specific for a variety of
pathogens or diseases including viruses, bacterial, fungal,
protozoan antigens, and/or tumor associated antigens. The antigen
can include T-lymphocyte-dependent proteins, haptenated proteins,
haptenated peptides, peptides, and the like as well as or
T-independent polysaccharides and haptens conjugated to
polysaccharides. The antigen can be polysaccharides, toxins, and
toxoids derived from pathogens. Examples of various antigens for
use with the present invention include antigens derived from or
including either whole or in part, viruses, such as, but not
restricted to: feline leukemia virus (FeLV), feline
immunodeficiency virus (FeIV), bovine LV, HIV-1, HIV-2, HTLV-1,
HTLV-II, rabies, measles, hepatitis B, hepatitis C, hoof and mouth
disease, influenza viruses, parainfluenza viruses, mumps virus,
adenoviruses, respiratory syncytial virus, Epstein-Barr virus,
rhinoviruses, polioviruses, coxsackieviruses, echoviruses, rubeola
virus, rubella virus, varicell-zoster virus, herpes viruses (human
and animal), herpes simplex virus, parvoviruses (human and animal),
cytomegalovirus, hepatitis viruses, human papillomavirus,
alphaviruses, flaviviruses, bunyaviruses, rabies virus,
arenaviruses, filoviruses, canine distemper virus, canine
contagious hepatitis virus, feline calicivirus, feline
rhinotracheitis virus, TGE virus (swine), cholera viruses, and
tetanus viruses.
[0028] Examples of bacterial antigens that can be used with the
present invention include, but are not restricted to, antigens
derived from or which include either whole or in part: anthrax,
diphtheria, Lyme disease, or tuberculosis; or protozoans, such as
Babeosis bovis or Plasmodium, Helicobacter pylori, Chlamydia
pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum,
Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus,
Streptococcus spp., Streptococcus pyogenes, Streptococcus
pneumoniae, Streptococcus viridans, Enterococcus faecalis,
Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis,
Salmonella spp., Salmonella typhi, Vibrio chlorea, Pasteurella
pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter
jejuni, Clostridium spp., Clostridium difficile, Mycobacterium
spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp.,
Borrelia burgdorferi, Leptospria spp., Hemophilus ducreyi,
Corynebacterium diphtheria, Bordetella pertussis, Bordetella
parapertussis, Bordetella bronchiseptica, hemophilus influenza,
Escherichia coli, Shigella spp., Erlichhia spp., and Rickettsia
spp. to name just a few examples.
[0029] Examples of fungal antigens that can be used with the
present invention include, but are not restricted to, antigens
derived from or which include either whole or in part: Aspergillus
fumigatus, Candida albicans, Histoplasma capsulatum, Coccidioides
immiti, or Blastomyces dermatiditis, to name just a few
examples.
[0030] Tumor associated antigens can be native, recombinant, or
synthetic immunogenic polypeptides or peptide fragments. Such tumor
associated antigens include, but are not limited to, killed tumor
cells and lysates thereof, MAGE-1 or MAGE-3 and peptide fragments
thereof, human chorionic gonadotropin (HCG) and peptide fragments
thereof, carcinoembryonic antigen (CEA) and peptide fragments
thereof, alpha fetoprotein (AFP) and peptide fragments thereof,
pancreatic oncofetal antigen and peptide fragments thereof, MUC-1
and peptide fragments thereof, CA 125, 15-3, 19-9, 549, 195 and
peptide fragments thereof, prostate-specific antigens (PSA) and
peptide fragments thereof, prostate-specific membrane antigen
(PSMA) and peptide fragments thereof, squamous cell carcinoma
antigen (SCCA) and peptide fragments thereof, ovarian cancer
antigen (OCA) and peptide fragments thereof, pancreas cancer
associated antigen (PaA) and peptide fragments thereof, Herl/neu
and peptide fragments thereof, gp-100 and peptide fragments
thereof, mutant K-ras proteins and peptide fragments thereof,
mutant p53 and peptide fragments thereof, truncated epidermal
growth factor receptor (EGFR), and chimeric protein
p210.sup.BCR-ABL.
[0031] The vaccine formulation can include the immunoadjuvant
component and antigen in an effective immunoadjuvant:antigen ratio
to elicit an immunogenic response. Preferable the
immunoadjuvant:antigen ratio in the present invention ranges
between about 20:80 wt % (wt:wt, adjuvant component:antigen
component) to about 50:50 wt %. More preferably, the vaccine
formulation can include the adjuvant component and the antigen
component in a weight ratio ranging between about 10:30 wt % to
about 10:20 wt %. The upper limit of phytol in the vaccine
formulation can be selected to be about 40 mg (50 .mu.l) and for
phytanol about 80-100 mg (100-125 .mu.l).
[0032] The vaccine formulation can include a carrier such as water,
preferably in buffered saline, or an oil-and-water emulsion, or
other physiologically suitable medium. The vaccine formulation can
include optional components in addition to the carrier. The
optional components can include surfactants, emulsifiers,
liposomes, cyclodextrin, and the like.
[0033] One of the other optional components can include an
emulsifying agent. Examples of emulsifying agents that can be
included in the present invention include naturally derived
materials such as gums from trees, vegetable protein, sugar-based
polymers such as alginates and cellulose specific examples
including: sorbital, mannitol, saccharose, glucose, and the like;
oxypolymers or polymers having a hydroxide or other hydrophilic
substituent on the carbon backbone having surfactant activity, for
example, povidone, polyethylene glycol, polyvinyl alcohol, and
glycol ether-based mono- and poly-functional compounds; long chain
fatty-acid-derived compounds such as ethers of fatty acids, esters
of fatty acids, glycerol, polyol lecithins, fatty acids, or
alcohols condensed with ethylene and/or propylene oxide. Liquid
emulsifiers are preferred.
[0034] The emulsifying agent can be included in the vaccine
formulation in a desired amount selected to provide an emission
stable for treatment of animals including humans. Preferably the
emulsifier is included in an amount selected to maintain a stable,
uniform, or homogeneous emulsion. In selected embodiments, the
selected amount can be between about 0.18 wt % and about 0.5 wt %
based upon the total weight of the vaccine formulation.
[0035] When the vaccine formulation includes a surfactant, the
surfactant can be included in an amount ranging between about 0.15
ml and 0.40 ml. Examples of surfactants include, but are not
restricted to, mannide monooleates, 1,4-sorbitan mono- and
triesters, polysorbate 80, polyoxyethylene derivatives of stearic
acid (sold under the trade name MYRJS.RTM.). The adjuvant component
can be selected to provide enhanced properties for the vaccine
formulation. Such enhanced properties can include reduced toxicity,
increased immunogenicity for the antigen, and longer retention at
the site of injection and/or within the body in general.
Consequently, a vaccine formulation can be prepared that includes
the antigen and the adjuvant component of the present invention.
This vaccine formulation can include the antigen in amounts lower
than that amount, which can be provided with the vaccine alone or
with the vaccine in combination with another antigen such as
aluminum hydroxide and still provide an equal or enhanced
immunogenic response. In preferred embodiments, the vaccine
formulation of the present invention can be formulated to include
the antigen in amounts ranging from about 10% to about 30% less
than that found in similar vaccine formulations formulated without
the phytol or phytol derivative as described herein. In selected
embodiments, the antigen can be included in the formulations of the
present invention in amounts between about 5-100 .mu.g.
[0036] As noted above the vaccine formulation of the present
invention can include the antigen directly bonded to the phytol,
phytanol or other phytol derivative (referred to below as "the
antigen phytol conjugate"). For example, the phytanol or other
phytol derivative can be directed bonded to a protein or a
carbohydrate. The vaccine formulation of this embodiment can
include the antigen-phytol conjugate in amounts effective to induce
an immunological response. More preferably the vaccine formulation
in unit dosage form can include the antigen-phytol conjugate in an
amount between about 4 to about 100 micrograms.
[0037] The formulation that includes the antigen-phytol conjugate
can be used in combination with a surfactant. Preferably the
surfactant and the antigen-phytol conjugate are combined in equal
volume amounts, optionally with a carrier or buffer solution.
Preferably a liquid vaccine formulation with a carrier includes the
surfactant in an amount between about 0.05 and about 0.1% (wt/v:
surfactant/carrier).
[0038] The adjuvant component of the present invention can be
prepared by combining the phytol or the phytol derivative in a
suitable medium for the vaccine. If the medium is physiological
saline, an emulsifier can be added to provide a homogeneous mixture
or suspension. Additional components can be added as desired. The
additional components can include an emulsifier, surface active
agent, phospholipids, and the like.
[0039] Non limiting examples of commercially available emulsifiers
and/or surfactants for use in the present invention include various
molecular weights of poly(ethylene glycol) (PEG 300, PEG 400, PEG
600, and PEG 800), sorbitan derivatives, and/or sorbitan esters of
fatty acids such as sorbitan monolaurate, sorbitan monopalmitate
sorbitan monostearate, sorbitan tristearate and sorbitan trioleate
and mixtures of these compounds Commercially available emulsifiers
useful in the present invention are sold under the trade names
TWEEN.RTM., ARLACEL.RTM., and SPAN.RTM., (available from ICI
America's, Inc., Wilmington, Del.).
[0040] The vaccine formulation according to the present invention
can be prepared by combining either the phytol, or the phytol
derivative and one or more antigens in a suitable carrier. The
order of addition is not critical to the practice of the present
invention. Alternatively, a liquid adjuvant component such as that
described above can be combined or admixed with a suitable antigen,
which may or may not be dissolved or suspended in a liquid medium.
Additional components such as surfactants, stabilizers, and alum
can also be combined with the antigen and adjuvant component.
Again, the order of addition is not critical to the practice of the
present invention. After the antigen and adjuvant or adjuvant
component and any additional components have been combined, the
resulting mixture is thoroughly stirred to ensure that the
resulting formulation is homogeneous.
[0041] The vaccine formulation can be administered by any medically
acceptable route including oral, nasal, mucosal, or injected
(preferably intramuscular injection).
[0042] For the purposes of promoting further understanding and
appreciation of the present invention and its advantages, the
following Examples are provided. It will be understood, however,
that these Examples are illustrative and not limiting in any
fashion.
EXAMPLE 1
Preparation of 2,3-Dihydrophytol (phytanol or H-phytol)
[0043] 2
[0044] To a solution of 1.00 g phytol in dry tetrahydrofuran was
added 0.10 g 5% palladium on carbon. The mixture was allowed to
stir 24 h under hydrogen at 1 atm. The catalyst was removed by
centrifugation and the solvent was then removed by rotary
evaporation. The resulting oil was purified by molecular
distillation at about 100.degree. C. and 0.1 mm Hg to give a
colorless oil: .sup.1H NMR.delta. (ppm) 0.90 (d, .about.15H), 1-1.8
(m, .about.25H), 3.7 (t, 2H).
[0045] Hydrogenation of phytol can also be accomplished using Raney
nickel and hydrogen to yield phytanol as referenced in Bendavid,
A., Burns, C. J., Leslie, D. F., Hashimoto, K., Ridley, D. D.,
Sandanayake, S., and Wieczorek L., J. Org. Chem. (2001), 66,
3709-3716; Kim, T., Chan, K. C, and Crooks, R. M., J. Am. Chem.
Soc. (1997) 119, 189; and Jellum, E., Eldjarn, L., and Try, K.,
Acta Chem. Scand. (1966), 20, 2335.
EXAMPLE 2
Preparation of Halogenated Phytol/Phytanol
[0046] Hologenated phytol can be prepared according the procedure
outlined below. The procedure shown below is a general one and can
be found in the above references, and in particular in Bendavid,
A., Burns, C. J., Leslie, D. F., Hashimoto, K., Ridley, D. D.,
Sandanayake, S., and Wieczorek, L., J. Org. Chem. (2001), 66,
3709-3716. 3
EXAMPLE 3
Preparation of Phytanyl Glucose/Phytanyl Mannose
[0047] Phytanyl glucose has been previously reported (1, 2). The
procedure used by Hato and coworkers (2) to make this compound in
moderate yield is the same procedure that they used to make some
similar acetals several years earlier (3). However, recent reports
(4, 5, 6) of the preparation of some similar lipidated carbohydrate
acetals appear to offer even better ways of making phytanyl
glucose. Hinguchi and coworkers (4) converted glucose acetate to a
bromide by treating with HBr and then converted this to an acetal
by treating it with a long-chain alcohol, AgClO.sub.4 and
AgCO.sub.3. It is envisioned that a procedure, as shown below, can
be used with phytanol. 4
[0048] Alternatively, a phytanyl glucose conjugate can be prepared
by adapting a procedure reported by Clausen and coworkers (glucose
acetate, ROH, BF.sub.3--OEt.sub.2, CH.sub.2Cl.sub.2) (5) or by the
procedure disclosed by Konstantinovic and coworkers (glucose
acetate, ROH, SnCl.sub.4) (6). A similar scheme can be adopted to
make phytanylated manose.
[0049] (1) Masakatsu, H.; Hiroyuki, N. Japanese patent number
JP2002226597
[0050] (2) Hato, M.; Minamikawa, H.; Salkar, R. A.; Matsutani, S.
Langmuir, 2002, 18, 3425-3429.
[0051] (3) Minamikawa, H.; Murakami, T.; Hato, M. Chem. Phys.
Lipids, 1994, 72, 111-118.
[0052] (4) Higuchi, R.; Mori, T.; Sugata, T.; Yamada, K.; Miyamoto,
T. European Journal of Organic Chemistry, 1999, 3175-3178.
[0053] (5) Clausen, M. H.; Jorgensen, M. R.; Thorsen, J.; Madsen,
R. J. Chem Soc., Perkin Trans. 1, 2001, 543-551.
[0054] (6) Konstantinovic, S.; Predojevic, J.; Gojkovic, S.;
Pavlovic, V. Indian Journal of Chemistry, 2003, 42B, 666-669.
EXAMPLE 4
Comparison of Phytol and Phytol Derivatives with Various
Commercially Available Adjuvants
[0055] Phytol-Induced Changes in BALB/c Mice
[0056] The efficacy and side effects (such as toxicity,
carcinogenic properties, and autoimmunogenic properties) of phytol
and reduced phytol or phytanol (H-phytol) as adjuvants were
compared with those of other known adjuvants that serve as the gold
standard. Groups of 8-12 week old and gender matched BALB/c mice
(n=21) were injected intraperitoneally with phytol (25-50
.mu.l=20-40 mg/ml injected vaccine formulation) to determine
toxicity. Similar studies were done with reduced phytol (phytanol)
using 11 BALB/c mice. LD50 for phytol is 40 mg/ml and for phytanol
>40 mg/ml. For comparison, equivalent concentrations/volumes of
other Freund's adjuvants, a mineral oil pristane and a control
(phosphate buffered saline) were tested for toxicity in terms of
splenomegaly and local granuloma. The results shown in FIG. 3A-3F
show comparable splenomegaly with phytol and CFA, but none with
phytanol. Phytol is milder than CFA in terms of splenomegaly and
granuloma. Phytanol (reduced phytol) shows no granuloma.
[0057] Carcinogenic Potential Assessment
[0058] Groups of BALB/c mice were treated with two intraperitoneal
injections (0.5 ml of pristane, and 50 .mu.l of phytol and Phytanol
each, 2 months apart). After 210 days, BALB/c mice injected with
only pristane showed appearance of ascites containing plasmacytoma
cells. The results in FIG. 4 clearly show that neither phytol nor
phytanol (reduced phytol) induces plasmacytoma, but it was observed
that pristane, a mineral oil component of many adjuvants, does
induce plasmacytomado.
[0059] Autoimmune Reactivity Assessment
[0060] In each set of experiments, groups of 5 mice were primed
with an environmental antigen phthalate (Lim and Ghosh, Immunology,
110: 482-492, 2003) as a conjugate (mg/ml) with keyhole limpet
hemocyanin (KLH) in different adjuvants as shown in FIG. 5. Phytol
and phytanol showed statistically insignificant anti-DNA response,
whereas all other adjuvants induced significant autoimmune
response.
[0061] Anti-Hapten Ab Response without Autoreactive Response
[0062] Experimental BALB/c mice (n=6) were grouped and primed
intraperitoneally with phthalate-KLH conjugates emulsified in each
adjuvant. Control groups of mice were immunized with phosphate
buffered saline (PBS). Boosters were given at 10-day intervals. The
mice were bled through retro-orbital veins five days after each
immunization. (reference describing this type of experiments:
So-Yon Lim and Swapan K. Ghosh, Immunology, 110: 482-492,
2003).
[0063] ELISA was performed to assess anti-phthalate humoral
responses on phthalate-BSA coated plates. The results in FIG. 6.
clearly indicate phytanol is better than commonly used CFA/IFA and
is as good as commercially available Ribi Adjuvant system (RAS)
which is primarily used for humoral antibody response.
[0064] Comparison of Antibody Isotype Induced
[0065] Sera from the above were also analyzed for different
antibody isotype using commercially available isotyping kit.
Results in FIG. 7 clearly showed the ability of phytol and phytanol
to induce IgG2a antibody isotype, which is the most effective in
activating complement and antibody-dependent cellular cytotoxic
mechanisms. This induction of IgG2a antibody is therapeutically
very useful for treatment of viral infection and cancer.
[0066] Induction of Tumor-Specific Cytotoxic (Killer) T Cells
[0067] A number of mice (BALB/c mice) were divided into four groups
of six mice each. Each group was treated with intraperitoneal
injections of one of six adjuvant preparations. The adjuvant
preparations were selected from IFA, phytol, and phytanol. A
control group was injected intraperitoneally with PBS. Five days
after the initial injections, each of the mice was injected with a
live tumor challenge (5.times.10.sup.6 of 2C3 B-cell lymphoma).
Splenocytes were obtained form the tumor-challenged mice at the
eighth day after the initial injections. The collected splenocytes
were stimulated with killed 2C3 cells in vitro for five days.
Effector cells were harvested from this preparation after five days
of incubation. The effector cells were used in the 51Cr-release
cytotoxic T lymphocyte (CTL) assay according to the procedure
published in Chakrabarti D. and Ghosh S. K.: Cellular Immunology
144: 445-464, 1996; Santra S. and Ghosh S. K.: cancer Immunol.
Immunotherpy, 44: 291-300, 1997; Laxmanan S., Stuart G. W. and
Ghosh S. K. Ghosh, Cancer Immunol. Immunotherapy, 50: 437-444,
2001; So-Yon Lim and Swapan K. Ghosh, Immunology, 110: 482-492,
2003.
[0068] From the above analysis it can be observed that phytol can
be used as an effective component in a wide variety of
pharmaceutical formulations and, in particular, shows promise as a
highly effective adjuvant for vaccines. The present invention
contemplates modifications as would occur to those skilled in the
art. It is also contemplated that compositions and formulations
embodied in the present invention can be altered or added to other
formulations as would occur to those skilled in the art without
departing from the spirit of the present invention.
[0069] Unless specifically identified to the contrary, all terms
used herein are used to include their normal and customary
terminology in the relevant fields of endeavor.
[0070] All publications, patents, and patent applications cited in
this specification are herein incorporated by reference as if each
individual publication, patent, or patent application was
specifically and individually indicated to be incorporated by
reference and set forth in its entirety herein. Further, any theory
of operation, proof, or finding stated herein is meant to further
enhance understanding of the present invention and is not intended
to make the scope of the present invention dependent upon such
theory, proof, or finding.
[0071] While the invention has been illustrated and described in
detail in the drawings and foregoing description, the same is
considered to be illustrative and not restrictive in character, it
is understood that only the preferred embodiments have been shown
and described and that all changes and modifications that come
within the spirit of the invention are desired to be protected.
* * * * *