U.S. patent application number 13/549329 was filed with the patent office on 2013-01-10 for metalloproteinase oligopeptides and their therapeutic use.
This patent application is currently assigned to MATTHIAS W. RATH. Invention is credited to Aleksandra Niedzwiecki, Matthias W. Rath, Waheed M. Roomi.
Application Number | 20130011423 13/549329 |
Document ID | / |
Family ID | 44455382 |
Filed Date | 2013-01-10 |
United States Patent
Application |
20130011423 |
Kind Code |
A1 |
Rath; Matthias W. ; et
al. |
January 10, 2013 |
METALLOPROTEINASE OLIGOPEPTIDES AND THEIR THERAPEUTIC USE
Abstract
The invention discloses identification and therapeutic use of
matrix metalloproteinase oligopeptides, SEQID 5-SEQID 21. These
oligopeptides are bound to antibodies to create an immune response
in the subject mammal against the matrix metalloproteinases of
various diseases. This is a means of therapeutic intervention
against the disease spread created by the matrix
metalloproteinases. Further use of these oligopeptide-antibody
responses can be extended to any and all diseases that use the
matrix metalloproteinases to aid in their pathogenicity.
Inventors: |
Rath; Matthias W.; (Aptos,
CA) ; Niedzwiecki; Aleksandra; (Aptos, CA) ;
Roomi; Waheed M.; (Sunnyvale, CA) |
Assignee: |
RATH; MATTHIAS W.
APTOS
CA
|
Family ID: |
44455382 |
Appl. No.: |
13/549329 |
Filed: |
July 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US11/42948 |
Jul 5, 2011 |
|
|
|
13549329 |
|
|
|
|
Current U.S.
Class: |
424/185.1 ;
424/94.67; 435/23 |
Current CPC
Class: |
C12N 9/64 20130101; C12N
9/6491 20130101; A61K 38/4886 20130101; A61K 39/0011 20130101; A61P
35/00 20180101; G02B 5/3058 20130101; G02B 5/1857 20130101; G02B
5/18 20130101; A61K 2039/6081 20130101; G02B 5/1861 20130101; C12N
9/50 20130101 |
Class at
Publication: |
424/185.1 ;
424/94.67; 435/23 |
International
Class: |
A61K 38/48 20060101
A61K038/48; C12Q 1/37 20060101 C12Q001/37; A61P 35/00 20060101
A61P035/00 |
Claims
1. A method, comprising: identifying an oligopeptide sequence for a
matrix metalloproteinase expressed in a specific disease;
synthesizing the oligopeptide sequence for the matrix
metalloproteinase, wherein the oligopeptide sequence of the matrix
metalloproteinase is at least one of SEQ ID 5 to 21 and combination
thereof; formulating a therapeutically effective amount of a
treatment composition using the oligopeptide sequence of the matrix
metalloproteinase; and administering the treatment composition of
the oligopeptide sequence to treat a mammal having the specific
disease exhibiting an over expression of the matrix
metalloproteinase.
2. The method of claim 1, wherein administering is injecting the
treatment composition as a vaccine to the mammal for treating the
specific disease.
3. The method of claim 2, wherein the specific disease is at least
one of neoplastic disease, inflammatory disease, coronary artery
disease, occlusive cardiovascular disease, degenerative disease and
infectious disease.
4. The method of claim 3, wherein the specific disease is
cancer.
5. The method of claim 1, further comprising: formulating the
therapeutically effective amount of the treatment composition using
a SEQ ID 5 to 21 oligopeptide sequence of the matrix
metalloproteinase; administering the treatment composition
comprising of at least one of the SEQ ID 5 to 21 and combination
thereof for treating the mammal; and generating an immune response
in a mammal.
6. The method of claim 1, wherein the therapeutically effective
amount is administered using at least one of a non-invasive
peroral, topical, enteral, transmucosal, targeted delivery,
sustained release delivery and parenteral method.
7. The method of claim 6, wherein the therapeutically effective
amount is the parenteral used as a vaccine.
8. A composition, comprising: a matrix metalloproteinase
oligopeptide; and a pharmaceutically acceptable excipient.
9. The composition of claim 8, further comprising: a carrier
protein; an additive to enhance the chemical stability; and an
adjuvant to increase the immunogenicity of the composition.
10. The composition of claim 8, wherein the matrix
metalloproteinase oligopeptide is at least one of SEQ ID 12 to 16
and combination thereof.
11. The composition of claim 8, wherein the matrix
metalloproteinase oligopeptide is at least one of linear and
circular in shape.
12. A method, comprising: measuring the level of expression of a
matrix metalloproteinase in a specific species having a specific
disease; and identifying an oligopeptide sequence for a matrix
metalloproteinase expressed in the specific disease to determine a
treatment method.
13. The method of claim 12, further comprising: synthesizing the
oligopeptide sequence of the matrix metalloproteinase; formulating
a treatment composition for producing a therapeutically effective
amount of the oligopeptide sequence of the matrix
metalloproteinase; and injecting the treatment composition as a
vaccine to the specific species for treating the specific disease
having an over expression of a matrix metalloproteinase.
14. The method of claim 13, further comprising: reducing a tumor
size by treating the specific species having the tumor, wherein the
specific disease is cancer, wherein the specific species are at
least one of a mammal and a non mammal.
15. The method of claim 14, wherein the treatment composition is
the injected directly into the tumor.
16. The method of claim 13, wherein the oligopeptide sequence for
the matrix metalloproteinase is at least one of a SEQ ID 5 to 21
and combination thereof.
17. The method of claim 14, further comprising: coupling the
sequence of matrix metalloproteinase with a hapten to enhance the
immune response as the vaccine.
18. The method of claim 17, further comprising; formulating the
therapeutically effective amount of the treatment composition using
a peptidomimetic sequence of the matrix metalloproteinase; and
administering the formula comprising of at least one of the matrix
metalloproteinase SEQ ID 5 to 21 and combination thereof as the
treatment composition for treating the specific species.
19. The method of claim 18, further comprising: reducing the
activity of at least one of a matrix metalloproteinase-2 and a
matrix metalloproteinase-9 using a vaccine comprising at least one
of a oligopeptide sequence of SEQ ID 5 to 21 and combination
thereof to induce an immune response.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The instant application is a continuation-in-part
application and claims priority to pending PCT application
PCT/US11/42948, filed on Jul. 5, 2011. The disclosure is hereby
incorporated by this reference in its entirety for all of its
teachings. This application contains sequence listing that has been
submitted as an ASCII file named RIPLLC018003CIP2_ST25, the date of
creation Jun. 19, 2012, and the size of the ASCII text file in
bytes is 4 kb.
FIELD OF TECHNOLOGY
[0002] This disclosure relates generally to designing and
synthesizing novel matrix metalloproteinase (MMP) oligopeptide
sequences to be used as therapeutic agents for treating
extracellular matrix related diseases. More specifically, this
disclosure relates to using the metalloproteinase oligopeptide from
various species as a vaccine, a pharmaceutical composition, a
therapeutic dose to treat extracellular matrix related diseases
that use MMP's.
BACKGROUND
[0003] Matrix Metalloproteinases are a family of zinc dependent
neutral endopeptidases that play an important role in tumor
angiogenesis, tissue remodeling, and cell migration. In cancer,
levels of some MMP's are abnormally elevated, enabling cancer cells
to degrade the extracellular matrix (ECM), invade the vascular
basement membrane, and metastasize to distant sites. A variety of
pathological conditions are associated with an increased activity
of metalloproteinases (MMP's), in particular MMP-9. These proteases
are able to digest collagen and other extracellular matrix (ECM)
proteins as a precondition for the spreading of the disease. Thus,
there is a need for a therapeutic agent to effectively block these
MMP's from digesting the ECM, thereby blocking the spread of cancer
and other diseases.
[0004] Prevention and treatment of metastasis represents the major
challenge in cancer therapy today. The current available treatments
are toxic, non-specific and unpredictable for ECM protein affected
diseases. There is a need for a therapeutic agent to effectively
block the MMP molecules from digesting the ECM, thereby preventing
ECM degradation and spreading of diseases.
SUMMARY
[0005] The current disclosure discloses a sequence and a
composition of MMP oligopeptide and a method of using the MMP
oligopeptide as a vaccine, a pharmaceutical composition, a
therapeutic dose and as a diagnostic for treating ECM related
diseases.
[0006] In one embodiment, the oligopeptide analogs for MMP-9
sequences were designed and synthesized. In another embodiment,
these oligopeptides were tested for generating immune response in
mouse for MMP sequences.
[0007] In one embodiment, the following oligopeptide sequences were
used to produce a vaccine. In another embodiment, a treatment dose
may be designed for a person suffering from cancer comprising of
SEQ ID 5-21 synthetic oligopeptide analogs.
TABLE-US-00001 TABLE 1 MMP-9 Synthetic Oligopeptide sequences
(Other Species): SEQ ID 5 Mouse MMP-9 A 5 D K D G K F G F SEQ ID 6
Bovine MMP-9 A 6 D A D R Q F G F SEQ ID 7 Rat MMP-9 A 7 D T D R K Y
G F SEQ ID 8 Carp MMP-9 A 8 D K D K I F G F SEQ ID 9 Pufferfish
MMP-9 A 9 D K D K K Y G F SEQ ID 10 Rabbit MMP-9 A 10 D T D R R F G
F SEQ ID 11 Mouse MMP-9 A 11 D K D G K F G F SEQ ID 12 Rat MMP-9 A
12 C H F P F T F E G R S Y L S C SEQ ID 13 Chicken MMP-9 A 13 C H F
P F I F E G R S Y S R C SEQ ID 14 European MMP-9 A 14 C H F P F L F
E G T S Y S S C Carp SEQ ID 15 Pufferfish MMP-9 A 15 C H F P F R F
Q N K P Y K H C SEQ ID 16 Flounder MMP-9 A 16 C H F P F T F E G K S
Y T S C SEQ ID 17 Bovine MMP-9 A 17 D Q D K L Y G F C P T R V D A
SEQ ID 18 Rat MMP-9 A 18 D K A D G F C P T R A D V T V SEQ ID 19
Mouse MMP-9 A 19 D Q D K L Y G F C P T R V D A SEQ ID 20 Carp MMP-9
A 20 D K K Y G F C P N R D T A V I SEQ ID 21 Rabbit MMP-9 A 21 D K
D K L Y G F C P T R A D S
[0008] In one embodiment, the sequence of oligopeptide may but is
not limited to have mutations, deletions and substitutions.
[0009] In one embodiment, the MMP-9 oligopeptide may be used as a
vaccine, a pharmaceutical composition, a therapeutic dose and as a
diagnostic tool. In another embodiment, all seventeen oligopeptides
may be combined to produce a vaccine.
[0010] The oligopeptide sequences, in one embodiment may be either
be linear or circular in design. In another embodiment, the
oligopeptide may be repeat of sequences.
[0011] In another embodiment, the oligopeptide may have either
haptens or polyglycans attached to them for efficient delivery.
[0012] In another embodiment, a method of immunizing a mammal, such
as rat, to raise antibodies for a specific MMP is disclosed. In one
embodiment, a selection of an oligopeptide suitable for raising
antigenicity is disclosed.
[0013] In one embodiment, the immunization of mammals may not be
limited to cancer but may include all ECM degradation based disease
treatment. In another embodiment, the vaccination may be done once
or repeatedly by measuring the antibodies specific to the
oligopeptide that was injected. In one embodiment, the specific
species may be one of a mammal and/or a non mammal.
[0014] In one embodiment, a composition for an oligopeptide as a
vaccine and a treatment dose comprising of oligopeptides comprising
of SEQ ID 5-21 individually or combination thereof.
[0015] In one embodiment, the therapeutically effective amount may
be rendered, but not limited to, as an injection. Other embodiments
may include peroral, topical, transmucosal, inhalation, targeted
delivery and sustained release formulations. The treatment dose may
comprise of therapeutically effective and pharmaceutically
acceptable combinations.
[0016] The composition, method, and treatment disclosed herein may
be implemented in any means for achieving various aspects, and may
be executed in a form suitable for the mammal. Other features will
be apparent from the accompanying drawings and from the detailed
description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Example embodiments are illustrated by way of example and
not limitation in the figures of the accompanying drawings, in
which like references indicate similar elements and in which:
[0018] FIG. 1 illustrates the prior art of MMP's digesting the ECM
during a disease state.
[0019] FIG. 2 the method of treating a mammal using the
vaccine.
[0020] FIGS. 3 and 3A show the immune response generated by the MMP
vaccine treatment.
[0021] Other features of the present embodiments will be apparent
from the accompanying drawings and from the detailed description
that follows.
DETAILED DESCRIPTION
[0022] Several sequences and methods for immunizing, treating
ECM-MMP related pathogenicity and raising an immune response by
vaccination are described herein. Although the present embodiments
have been described with reference to specific example embodiments,
it will be evident that various modifications and changes may be
made to these embodiments without departing from the broader spirit
and scope of the various embodiments.
[0023] Cancer cells produce higher levels of matrix
metalloproteinases (MMP's), particularly MMP-9. These enzymes are
able to digest the extra cellular matrix (ECM) connective tissue
surrounding the cancerous cells. MMP's bind to ECM via specific
binding sites. Blocking these binding sites in the MMP's prevents
the MMP's from binding to ECM. Inhibition of ECM destruction
prevents the cancer progression and leads to tumor size reduction.
In the current disclosure several potential binding sites were
identified within MMP-9. FIG. 1 describes the cancerous cells 110
producing MMP's 120 (step 1). The MMP's 120 bind to the specific
binding sites at the ECM 130 (step 2). Step 3 in FIG. 1 shows the
MMP's 120 digesting the ECM 140.
[0024] FIG. 2 explains the current disclosure in steps of vaccine
production and immunization 200. Identification and design of the
MMP's are carried out 210 using the MMP sequences. Oligopeptides
are synthesized 220 and represented by synthetic oligopeptides 240.
The formulate oligopeptides for vaccination are 222. The antibodies
are produced as shown in 226. The mouse 242 is immunized 224 using
vaccine with syringe 240. The oligopeptides may be of specific
length. Several permutations and combinations of the sequences were
tested. The instant disclosure sequences are shown in para [0009]
as MMP-9 SEQ ID #5-21. Prior to the selection of these seventeen
sequences as potential oligopeptides, several potential binding
sites for MMP-9 were identified. Finally a total of seventeen
oligopeptides were synthesized and tested on mouse. The MMP-9 5 to
21 was synthesized in a linear and circular format. Many
modifications for these sequences were also done in one embodiment.
The modifications were substituting one or more amino acid residues
at N-terminal, C-terminal and both C and N terminals, substitution
of amino acid residues based on similar charge and polarity,
without consideration of charge and polarity, omitting of amino
acids in C and N terminal, omitting only in C-terminal and only in
N-terminal.
[0025] In another embodiment, substitution and omission may be
carried out simultaneously. The oligopeptides may be further
modified by repeating the sequences and combining more than one SEQ
ID 5-21 for producing and formulating a vaccine. The peptidomimetic
to the MMP's may be used to block the binding site of an
overexpressed MMP in a specific disease.
[0026] In one embodiment, the oligopeptide may be used as feedback
regulators to specifically prevent or reduce the synthesis rate of
MMP-9 productions at the cellular level. In one embodiment process
of blocking and inhibition of ECM destruction by antigens produced
due to vaccination of mouse.
Preparation of Peptide Solutions for Immunization
[0027] The design of the experiment was done such that Peptides are
dissolved at concentration 1.1 mg/ml. Conjugate Streptavidin-HRP as
a carrier protein was dissolved in PBS at concentration 0.8 mg/ml.
Peptide solution was mixed with conjugate Streptavidin-HRP to
achieve standard final concentrations for peptides and
conjugate.
[0028] Conjugate Streptavidin-PolyHRP20 (#SP20C) as a carrier
protein was purchased from SDT (Germany), dialysis tubing cellulose
membrane D9777-100FT, Sigma (St. Louis, Mo.), glass vials ISO
8362-1 2R-CL-1 (Medical Glass, Bratislava, Slovakia) or PP Costar
Microcentrifuge Tube (Cat. #3621, Corning Inc., USA), urea and
salts were obtained from Fluka (Schweiz). All the reagents used
were of analytical grade. All solutions were prepared using pyrogen
free milliQ grade water. "SP-35" from gp41 env HIV-1 was used as a
Reference peptide with biotin having the following sequence:
H-Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp--
Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr-Ala-Val-Pro-Trp-Asn-Ala-Ser-OH.
[0029] Testing of Peptide Solubility:
[0030] Data for peptide solubility are represented in Table 1 and
2. Peptides SEQ ID 5-21 was dissolved in 8M Urea to a final
concentration of 1.1 mg/ml.
TABLE-US-00002 TABLE 1 MMP 9 Peptides from various species:
Solubility test Peptide Weight, mg 8M Urea, ml Solubility SEQ ID 5
0.39 0.43 Soluble SEQ ID 6 0.37 0.405 Soluble SEQ ID 7 0.69 0.76
Soluble SEQ ID 8 0.38 0.42 Soluble SEQ ID 9 0.62 0.68 Soluble SEQ
ID 10 0.56 0.515 Soluble SEQ ID 11 0.53 0.585 Soluble SEQ ID 12
0.43 0.475 Insoluble SEQ ID 13 0.64 0.705 Soluble SEQ ID 14 0.44
0.485 Soluble SEQ ID 15 0.51 0.56 Soluble SEQ ID 16 0.5 0.55
Soluble SEQ ID 17 0.58 0.64 Soluble SEQ ID 18 0.57 0.625 Soluble
SEQ ID 19 0.65 0.715 Soluble SEQ ID 20 0.48 0.53 Soluble SEQ ID 21
0.63 0.695 Soluble
[0031] MMP 9 Peptide SEQ ID 12 was insoluble in 8M Urea hence equal
volume of 0.1 M Na-carbonate buffer, pH 9.5 were added to dissolve
it in a solution as shown in Table 2.
TABLE-US-00003 TABLE 2 Additional Peptide Weight, mg 8M Urea, ml
solution, ml Solubility SEQID5 4.67 1.46 Soluble SEQID6 4.63 1.445
Soluble SEQID7 4.82 1.505 Soluble SEQID8 4.29 1.34 Soluble SEQID9
4.85 1.515 Soluble SEQID10 4.44 1.39 Soluble SEQID11 4.63 1.445
Soluble SEQID12 4.54 0.71 0.71 ml 0.1M Soluble Na-carbonate buffer,
pH 9.5 SEQID13 4.75 1.485 Soluble SEQID14 4.54 1.42 Soluble SEQID15
4.56 1.425 Soluble SEQID16 4.75 1.485 Soluble SEQID17 4.27 1.335
Soluble SEQID18 4.32 1.35 Soluble SEQID19 4.54 1.42 Soluble SEQID20
4.53 1.415 Soluble SEQID21 4.54 1.42 Soluble SP35 4.29 1.34
Soluble
[0032] Preparation of Conjugate Streptavidin-PolyHRP20 as a carrier
protein. Conventionally the conjugate Streptavidin-PolyHRP20
(Str-HRP 1 mg/ml) comes as solution containing 50% (v/v) glycerol.
For removing the glycerol, Str-HRP was dialyzed against Phosphate
buffer saline (PBS). Volume of conjugate increases after dialysis
and it has to be concentrated to a final volume containing 1.25
(0.8 mg/ml) concentration.
[0033] Preparation of conjugate peptide with carrier protein.
[0034] Peptides were dissolved in appropriate volume 8M Urea and
additional solution. Final concentration of peptide-Str-HRP
solution was 0.8 mg/ml and 0.6 mg/ml for peptide and Str-HRP,
respectively. After dissolving, 4 aliquotes in 0.2 ml portions were
taken from each peptide solution, mixed with 0.6 ml Str-HRP and
incubated over night at +4.degree. C. Conjugate peptide with
carrier protein (Str-HRP) was frozen and stored at -20.degree. C.
until further use. Final concentration of urea in peptide-Str-HRP
solution was 2M for all peptides.
Immunization
[0035] BALB/c female mouse, complete Freund's adjuvant (Calbiochem,
USA), incomplete Freund's adjuvant (Calbiochem, USA), 2 ml syringe
22G.times.11/2'' (BKMI, R. Korea), PP Costar Microcentrifuge Tube
(Cat. #3621, Corning Inc., USA), Vortex Vibrofix VF1 (IKA-Werk,
Germany), GP Centrifuge (Beckman, USA) were used as materials.
[0036] The pattern for immunization was as follows:
Day 0: Immunization with complete Freund's adjuvant Day 7: Booster
with incomplete Freund's adjuvant Day 14: Booster with incomplete
Freund's adjuvant Day 28: Booster with incomplete Freund's adjuvant
Day 38: Terminal bleed
[0037] Method of Immunization:
[0038] Frozen 0.8 ml aliquotes of peptide+Str-HRP conjugate were
thawed at room temperature (RT) and mixed with 0.8 ml of
appropriate adjuvant. Adjuvant was added and mixed by vortexing
immediately before injections were given. Immunization was done by
using intra peritoneal injections with 100 .mu.g peptide per animal
in final volume 250 .mu.l of 1:1 (v:v) peptide+Str-HRP:adjuvant.
Five animals were used per peptide.
[0039] Preparation of Serum:
[0040] At 38 days, the mouse was bled. Blood was collected in 2-ml
microcentrifuge tube and the blood was allowed to clot at room
temperature for 1 hour. Centrifugation using the microcentrifuge
tube with the clot inside was done for 15 min at 2500 g and serum
was collected. Volume for each sample was no less than 400 .mu.l.
The serum was stored at -20.degree. C.
[0041] Testing Immune Response to Individual Peptide:
[0042] Determination of mouse antibodies to peptide based on
indirect solid-phase immunoenzymatic assay with avidin on
solid-phase was performed. Materials used were 96-well polystyrene
plates high binding (#9018 Costar, USA), all solution for ELISA:
sample diluent (10 mM sodium phosphate, 500 mM NaCl, 0.5% BSA,
0.05% Tween-20, pH 7.4), conjugate diluent (10 mM sodium phosphate,
150 mM NaCl, 0.5% BSA, 0.05% Tween-20, pH 7.4), wash fluid (10 mM
sodium phosphate, 300 mM NaCl, 0.05% Tween-20, pH 7.4), substrate
buffer (50 mM sodium citrate and hydrogen peroxide, pH 5.0), TMB
solution (3,3',5,5'-tetramethylbenzidine), stop solution (2M
sulphuric acid) were taken from EIA Kit for detection of antibody
to HIV "Peptoscreen-2" (Amercard Ltd, Russia), conjugate of rabbit
antibody to mouse IgG with HRPO (custom made), avidin from egg
white (Imtek, Russia). Negative controls of 5 sera were pooled from
the nonimmune BALB/c female mouse. Dispensing of solutions with
Finnpipette digital adjustable volume pipettes 0.5-10 .mu.l, 5-40
.mu.l, 20-200 .mu.l, 200-1000 .mu.l, 1-5 ml and 12-channel pipettes
5-50 .mu.l, 50-200 .mu.l. Plate washer ZLE201 (Amersham Inc., UK),
incubator at 37.degree. C.--Imperial II (Lab-Line Instruments Inc.,
USA), EIA plate reader--Luminometer-Photometer LM01A (Immunotech,
Beckman Coulter Company, USA).
[0043] Test Procedure:
[0044] Peptides for binding on the avidin-coated plate were taken
from test-solution for determining the solubility of peptides
(chapter 1.3., table 1 and 2) and were dissolved up to 2 mM in
sample diluents immediately before the test procedure as shown in
Table 3.
TABLE-US-00004 TABLE 3 .mu.l peptides solution 1.1 mg/ml Peptide
MolWeight 2 mM mg/ml on 10 ml sample diluent SEQID5 1254.44 2.509
23 SEQID6 1294.44 2.589 23.5 SEQID7 1340.51 2.681 24.5 SEQID8
1308.55 2.617 24 SEQID9 1339.57 2.679 24.5 SEQID10 1352.52 2.705
24.5 SEQID11 1254.44 2.509 23 SEQID12 2133.48 4.267 39 SEQID13
2188.56 4.377 40 SEQID14 2064.37 4.129 37.5 SEQID15 2291.73 4.583
42 SEQID16 2093.41 4.187 38 SEQID17 2067.36 4.135 37.5 SEQID18
1934.21 3.868 35 SEQID19 2067.36 4.135 37.5 SEQID20 2066.41 4.133
37.5 SEQID21 2055.34 4.111 37.5 SP35 3702.6 7.405 67
[0045] EIA plates were coated by adding to a well containing 100
.mu.l of avidin dissolved 10 .mu.g/ml in 50 mM carbonate buffer, pH
9.5 and incubated for 20 h at 20.degree. C. The plates were washed
4 times with wash fluid. Peptides 2 mM, 100 ml/well in sample
diluent and incubated 60 min at 37.degree. C. Control wells contain
avidin. The plates were washed 4 times with wash fluid. Serum from
each mouse and negative control were diluted 1:100, 1:1000 and
1:10000 in sample diluents and were added to the wells, coated by
the corresponding peptide (100 .mu.l per well) and incubated for 1
h at 37.degree. C. The plates were washed 4 times with wash fluid.
Conjugates of rabbit anti-mouse IgG antibody with HRPO (dilution of
1:3000 in conjugate diluent) were added to the wells (100 .mu.l per
well). The plates were incubated for 0.5 h at 37.degree. C. The
plates were washed 4 times with wash fluid. 100 .mu.l of freshly
prepared substrate solution (1 v TMB solution+7 v substrate buffer)
were added to each well, and the plates were left at room
temperature for 15 minutes in a dark place. A blue color should
develop in wells containing positive samples. 100 .mu.l stop
solution was added to each well in the same sequence as the
addition of substrate solution. This causes the blue color to
change to yellow. Plates were read within 50 minutes at 450 nm
(A.sub.450) using a plate reader. The absorbance of each plate was
read as well.
[0046] Testing results of immune response to individual peptide by
immunoenzymatic assay of individual antiserum are presented as
signal A.sub.450 in addendum table 4. The results are as follows:
Nmouse--Negative control, Mouse #--signal of each individual
antiserum, mean--average of signal of A.sub.450 for each dilution,
mean-N--difference between mean and Nmouse, Avidin(A)--signal on
avidin and mean-A--difference between mean and avidin. Analysis of
the results shows that the most active of immune response were
obtained with peptides SEQID #15 and 13.
TABLE-US-00005 TABLE 4 OD 450 Mean values (n = 3-5) for PEPTIDE
dilution 1:10,000 RANK SEQID5 0.845 5 SEQID6 0.096 17 SEQID7 1.610
4 SEQID8 0.097 16 SEQID9 0.131 15 SEQID10 9.212 3 SEQID11 0.183 12
SEQID12 0.661 9 SEQID13 12.882 2 SEQID14 0.394 11 SEQID15 21.81 1
SEQID16 0.823 6 SEQID17 0.780 8 SEQID18 0.138 13 SEQID19 0.818 7
SEQID20 0.138 14 SEQID21 0.411 10 SP35 reference peptide 18.135
(positive control)
[0047] Conclusion:
[0048] All 17 peptides tested fall in three groups. Group of
relatively strong immunogenicity A.sub.450>1,0 for dilution
1:1000 (SEQ ID #A7, 10, 13, 15). Group of intermediate
immunogenicity A.sub.450>2,0 for dilution 1:100 and
A.sub.450<1,0 for dilution 1:1000 (SEQ ID #5, 12, 14, 16, 17,
19, 21). Group of weak immunogenicity A.sub.450<2,0 for dilution
1:100 remaining 6 peptides.
[0049] The oligopeptide therapeutically effective amount may be
administered to the mammal in many different ways and may not be
limited to injections. The various methods of administration are
well known in the art and some of the methods are described
below.
[0050] A "specific species" to be treated by the subject method may
mean a human or non-human animal, such as mouse, farm animals,
primates and vertebrates.
[0051] The specific diseases that would be target diseases for a
treatment using MMP oligopeptide sequences and/or peptidomimetic
are neoplastic diseases, inflammatory diseases, coronary artery
diseases, occlusive cardiovascular diseases, degenerative diseases
and infectious diseases. Some examples of neoplastic diseases may
be, but not limited to, cancer, lymphoma, leukemia, and brain
tumor. Some examples of inflammatory diseases may be, but not
limited to, arthritis, asthma, atherosclerosis, Crohn's disease,
colitis, dermatitis, lupus erythematous etc. Some examples of
infectious diseases may include, but not limited to, are bacterial,
viral, fungal, mycoplasmal, certain genetic diseases and other
infections. In the instant application, signal oligopeptides within
the selective MMP protein that mediate MMP protein's key
pathological function, namely the digestion of the connective
tissue, which is a precondition for cancer cells to migrate and
metastasize were identified. The oligopeptides were synthesized,
the animals were injected and antibodies were raised. The tumor
size was significantly reduced using these oligopeptide raised
vaccines. The degree of efficacy can be seen by the titer level
caused by MMP oligopeptide vaccine which proves the vaccines were
very effective.
[0052] One of the conventional methods of choice to increase the
natural antigenicity of the oligopeptide sequence(s) of a given
protein is a slight alteration in the amino acid sequence within
the given oligopeptide, i.e. by substitutions, deletions,
insertions etc. of individual amino acids. The SEQ ID 5-21 were
identified and designed in such a way that it matches the
hydrophobicity, hydrophylicity and the electrical charge of the
amino acids oligopeptide sequences of SEQ ID A1-A3 (MMP) as shown
in the previous application. It also maintains the signal
characteristics and functionality as an epitope. Since these
oligopeptides are very similar to the SEQ ID A1-A3 (MMP) that may
be used as vaccine for the reduction of tumor growth. The instant
application presents these sequences as a potential candidate for
vaccine development to treat cancer. The SEQ ID's 5-21 may enhance
the therapeutic efficacy for cross species immunization by
enhancing natural antigenicity irrespective of whether the
adjuvants are used.
[0053] Drug formulations suitable for these administration routes
can be produced by adding one or more pharmacologically acceptable
carriers to the agent and then treating the mixture through a
routine process known to those skilled in the art. The mode of
administration includes, but not limited to, are non-invasive
peroral, topical (example transdermal), enteral, transmucosal,
targeted delivery, sustained release delivery, delayed release,
pulsed release and parenteral methods. Peroral administration may
be administered both in liquid and dry state.
[0054] Formulations suitable for oral administration may be in the
form of capsules, cachets, pills, tablets, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or
non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia), each
containing a predetermined amount of a subject composition as an
active ingredient. Subject compositions may also be administered as
a bolus, electuary, or paste.
[0055] When an oral solid drug product is prepared, oligopeptide
sequence of MMP and/or a peptidomimetic of the MMP's is mixed with
an excipient (and, if necessary, one or more additives such as a
binder, a disintegrant, a lubricant, a coloring agent, a sweetening
agent, and a flavoring agent), and the resultant mixture is
processed through a routine method, to thereby produce an oral
solid drug product such as tablets, coated tablets, granules,
powder, or capsules. Additives may be those generally employed in
the art. Examples of the excipient include lactate, sucrose, sodium
chloride, glucose, starch, calcium carbonate, kaolin,
microcrystalline cellulose, and silicic acid; examples of the
binder include water, ethanol, propanol, simple syrup, glucose
solution, starch solution, liquefied gelatin,
carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl
starch, methyl cellulose, ethyl cellulose, shellac, calcium
phosphate, and polyvinyl pyrrolidone; examples of the disintegrant
include dried starch, sodium arginate, powdered agar, sodium
hydrogencarbonate, calcium carbonate, sodium lauryl sulfate,
monoglyceryl stearate, and lactose; examples of the lubricant
include purified talc, stearic acid salts, borax, and polyethylene
glycol; and examples of the sweetening agent include sucrose,
orange peel, citric acid, and tartaric acid.
[0056] When a liquid drug product for oral administration is
prepared, oligopeptide sequence of MMP and/or a peptidomimetic of
MMP's is mixed with an additive such as a sweetening agent, a
buffer, a stabilizer, or a flavoring agent, and the resultant
mixture is processed through a routine method, to thereby produce
an orally administered liquid drug product such as an internal
solution medicine, syrup, or elixir. Examples of the sweetening
agent include vanillin; examples of the buffer include sodium
citrate; and examples of the stabilizer include tragacanth, acacia,
and gelatin.
[0057] For purposes of transdermal (e.g., topical) administration,
dilute sterile, aqueous or partially aqueous solutions (usually in
about 0.1% to 5% concentration), otherwise similar to the above
parenteral solutions, may be prepared.
[0058] Formulations for rectal or vaginal administration may be
presented as a suppository, which may be prepared by mixing a
subject composition with one or more suitable non-irritating
carriers comprising, for example, cocoa butter, polyethylene
glycol, a suppository wax, or a salicylate, and which is solid at
room temperature, but liquid at body temperature and, therefore,
will melt in the appropriate body cavity and release the
encapsulated compound(s) and composition(s). Formulations which are
suitable for vaginal administration also include pessaries,
tampons, creams, gels, pastes, foams, or spray formulations
containing such carriers as are known in the art to be
appropriate.
[0059] A targeted release portion can be added to the extended
release system by means of either applying an immediate release
layer on top of the extended release core; using coating or
compression processes or in a multiple unit system such as a
capsule containing extended and immediate release beads.
[0060] When used with respect to a pharmaceutical composition or
other material, the term "sustained release" is art-recognized. For
example, a therapeutic composition which releases a substance over
time may exhibit sustained release characteristics, in contrast to
a bolus type administration in which the entire amount of the
substance is made biologically available at one time. For example,
in particular embodiments, upon contact with body fluids including
blood, spinal fluid, mucus secretions, lymph or the like, one or
more of the pharmaceutically acceptable excipients may undergo
gradual or delayed degradation (e.g., through hydrolysis) with
concomitant release of any material incorporated therein, e.g., an
therapeutic and/or biologically active salt and/or composition, for
a sustained or extended period (as compared to the release from a
bolus). This release may result in prolonged delivery of
therapeutically effective amounts of any of the therapeutic agents
disclosed herein.
[0061] Current efforts in the area of drug delivery include the
development of targeted delivery in which the drug is only active
in the target area of the body (for example, in cancerous tissues)
and sustained release formulations in which the drug is released
over a period of time in a controlled manner from a formulation.
Types of sustained release formulations include liposomes, drug
loaded biodegradable microspheres and drug polymer conjugates.
[0062] Delayed release dosage formulations are created by coating a
solid dosage form with a film of a polymer which is insoluble in
the acid environment of the stomach, but soluble in the neutral
environment of the small intestines. The delayed release dosage
units can be prepared, for example, by coating a drug or a
drug-containing composition with a selected coating material. The
drug-containing composition may be a tablet for incorporation into
a capsule, a tablet for use as an inner core in a "coated core"
dosage form, or a plurality of drug-containing beads, particles or
granules, for incorporation into either a tablet or capsule.
Preferred coating materials include bioerodible, gradually
hydrolyzable, gradually water-soluble, and/or enzymatically
degradable polymers, and may be conventional "enteric" polymers.
Enteric polymers, as will be appreciated by those skilled in the
art, become soluble in the higher pH environment of the lower
gastrointestinal tract or slowly erode as the dosage form passes
through the gastrointestinal tract, while enzymatically degradable
polymers are degraded by bacterial enzymes present in the lower
gastrointestinal tract, particularly in the colon. Alternatively, a
delayed release tablet may be formulated by dispersing tire drug
within a matrix of a suitable material such as a hydrophilic
polymer or a fatty compound. Suitable hydrophilic polymers include,
but are not limited to, polymers or copolymers of cellulose,
cellulose ester, acrylic acid, methacrylic acid, methyl acrylate,
ethyl acrylate, and vinyl or enzymatically degradable polymers or
copolymers as described above. These hydrophilic polymers are
particularly useful for providing a delayed release matrix. Fatty
compounds for use as a matrix material include, but are not limited
to, waxes (e.g. carnauba wax) and glycerol tristearate. Once the
active ingredient is mixed with the matrix material, the mixture
can be compressed into tablets.
[0063] A pulsed release-dosage is one that mimics a multiple dosing
profile without repeated dosing and typically allows at least a
twofold reduction in dosing frequency as compared to the drug
presented as a conventional dosage form (e.g., as a solution or
prompt drug-releasing, conventional solid dosage form). A pulsed
release profile is characterized by a time period of no release
(lag time) or reduced release followed by rapid drug release.
[0064] The phrases "parenteral administration" and "administered
parenterally" as used herein refer to modes of administration other
than enteral and topical administration, such as injections, and
include without limitation intravenous, intramuscular,
intrapleural, intravascular, intrapericardial, intra-arterial,
intrathecal, intracapsular, intraorbital, intracardiac,
intradennal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intra-articular, subcapsular, subarachnoid,
intraspinal and intrasternal injection and infusion.
[0065] Certain pharmaceutical compositions disclosed herein
suitable for parenteral administration comprise one or more subject
compositions in combination with one or more pharmaceutically
acceptable sterile, isotonic, aqueous, or non-aqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic within
the blood of the intended recipient or suspending or thickening
agents.
[0066] When an injection product is prepared, oligopeptide sequence
of MMP and/or a peptidomimetic of MMP's is mixed with an additive
such as a pH regulator, a buffer, a stabilizer, an isotonicity
agent, or a local anesthetic, and the resultant mixture is
processed through a routine method, to thereby produce an injection
for subcutaneous injection, intramuscular injection, or intravenous
injection. Examples of the pH regulator or buffer include sodium
citrate, sodium acetate, and sodium phosphate; examples of the
stabilizer include sodium pyrosulfite, EDTA, thioglycollic acid,
and thiolactic acid; examples of the local anesthetic include
procaine hydrochloride and lidocaine hydrochloride; and examples of
the isotonicity agent include sodium chloride and glucose.
[0067] Adjuvants are used to enhance the immune response. Various
types of adjuvants are available. Haptens are used as adjuvants in
this disclosure. Freund's adjuvants may also be used to produce
water-in-oil emulsions of immunogens. Antigens in water-in-oil
emulsions stimulate high and long-lasting antibody responses which
can be attributed to the slow release of antigen. Antigens
(preferably in saline) are typically mixed with an equal volume of
the adjuvant to form an emulsion.
[0068] The phrase "pharmaceutically acceptable" is art-recognized.
In certain embodiments, the term includes compositions, polymers
and other materials and/or dosage forms which are within the scope
of sound medical judgment, suitable for use in contact with the
tissues of mammals, human beings and animals without excessive
toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk
ratio.
[0069] The phrase "pharmaceutically acceptable carrier" is
art-recognized, and includes, for example, pharmaceutically
acceptable materials, compositions or vehicles, such as a liquid or
solid filler, diluent, solvent or encapsulating material involved
in carrying or transporting any subject composition, from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of a subject composition and
not injurious to the patient. In certain embodiments, a
pharmaceutically acceptable carrier is non-pyrogenic. Some examples
of materials which may serve as pharmaceutically acceptable
carriers include: (1) sugars, such as lactose, glucose and sucrose;
(2) starches, such as corn starch and potato starch; (3) cellulose,
and its derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt;
(6) gelatin; (7) talc; (8) cocoa butter and suppository waxes; (9)
oils, such as peanut oil, cottonseed oil, sunflower oil, sesame
oil, olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0070] In certain embodiments, the pharmaceutical compositions
described herein are formulated in a manner such that said
compositions will be delivered to a mammal in a therapeutically
effective amount, as part of a prophylactic, preventive or
therapeutic treatment.
[0071] In certain embodiments, the dosage of the oligopeptide
compositions, which may be referred as therapeutic composition
provided herein may be determined by reference to the plasma
concentrations of the therapeutic composition or other encapsulated
materials. For example, the blood samples may be tested for their
immune response to their corresponding oligopeptides.
[0072] The therapeutic compositions provided by this application
may be administered to a subject in need of treatment by a variety
of conventional routes of administration, including orally,
topically, parenterally, e.g., intravenously, subcutaneously or
intramedullary. Further, the therapeutic compositions may be
administered intranasally, as a rectal suppository, or using a
"flash" formulation, i.e., allowing the medication to dissolve in
the mouth without the need to use water. Furthermore, the
compositions may be administered to a subject in need of treatment
by controlled release dosage forms, site specific drug delivery,
transdermal drug delivery, patch (active/passive) mediated drug
delivery, by stereotactic injection, or in nanoparticles.
[0073] Expressed in terms of concentration, an active ingredient
can be present in the therapeutic compositions of the present
invention for localized use about the cutis, intranasally,
pharyngolaryngeally, bronchially, intravaginally, rectally, or
ocularly.
[0074] For use as aerosols, the active ingredients can be packaged
in a pressurized aerosol container together with a gaseous or
liquefied propellant, for example, dichlorodifluoromethane, carbon
dioxide, nitrogen, propane, and the like, with the usual adjuvants
such as cosolvents and wetting agents, as may be necessary or
desirable.
[0075] The most common routes of administration also include the
preferred transmucosal (nasal, buccal/sublingual, vaginal, ocular
and rectal) and inhalation routes.
[0076] In addition, in certain embodiments, subject compositions of
the present application maybe lyophilized or subjected to another
appropriate drying technique such as spray drying. The subject
compositions may be administered once, or may be divided into a
number of smaller doses to be administered at varying intervals of
time, depending in part on the release rate of the compositions and
the desired dosage.
[0077] Formulations useful in the methods provided herein include
those suitable for oral, nasal, topical (including buccal and
sublingual), rectal, vaginal, aerosol and/or parenteral
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any methods well known in
the art of pharmacy. The amount of a subject composition which may
be combined with a carrier material to produce a single dose may
vary depending upon the subject being treated, and the particular
mode of administration.
[0078] The therapeutically acceptable amount described herein may
be administered in inhalant or aerosol formulations. The inhalant
or aerosol formulations may comprise one or more agents, such as
adjuvants, diagnostic agents, imaging agents, or therapeutic agents
useful in inhalation therapy. The final aerosol formulation may for
example contain 0.005-90% w/w, for instance 0.005-50%, 0.005-5%
w/w, or 0.01-1.0% w/w, of medicament relative to the total weight
of the formulation.
[0079] Examples of suitable aqueous and non-aqueous carriers which
may be employed in the pharmaceutical compositions include water,
ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable
oils, such as olive oil, and injectable organic esters, such as
ethyl oleate. Proper fluidity may be maintained, for example, by
the use of coating materials, such as lecithin, by the maintenance
of the required particle size in the case of dispersions, and by
the use of surfactants. The therapeutic acceptable dosage may be
combined with other drugs and may be treated as a combination
drug.
[0080] In addition, it will be appreciated that the various
sequences, immunization processes, and methods of treatment
disclosed herein may be embodied using means for achieving the
various combinations of therapeutic dosage and delivery methods to
treat a specific disease such as cancer. Accordingly, the
specification and drawings are to be regarded in an illustrative
rather than a restrictive sense.
Sequence CWU 1
1
1718PRTArtificial SequenceMouse Synthetic OligoPeptide 1Asp Lys Asp
Gly Lys Phe Gly Phe1 528PRTArtificial SequenceBovine Synthetic
OligoPeptide 2Asp Ala Asp Arg Gln Phe Gly Phe1 538PRTArtificial
SequenceRat Synthetic OligoPeptide 3Asp Thr Asp Arg Lys Tyr Gly
Phe1 548PRTArtificial SequenceCARP Synthetic OilgoPeptide 4Asp Lys
Asp Lys Ile Phe Gly Phe1 558PRTArtificial SequencePufferfish
Synthetic OligoPeptide 5Asp Lys Asp Lys Lys Tyr Gly Phe1
568PRTArtificial SequenceRabbit Synthetic OligoPeptide 6Asp Thr Asp
Arg Arg Phe Gly Phe1 578PRTArtificial SequenceMouse Synthetic
OligoPeptide 7Asp Lys Asp Gly Lys Phe Gly Phe1 5815PRTArtificial
SequenceRat Synthetic OligoPeptide 8Cys His Phe Pro Phe Thr Phe Glu
Gly Arg Ser Tyr Leu Ser Cys1 5 10 15915PRTArtificial
SequenceChicken Synthetic OligoPeptide 9Cys His Phe Pro Phe Ile Phe
Glu Gly Arg Ser Tyr Ser Arg Cys1 5 10 151015PRTArtificial
SequenceEUROPEAN CARP Synthetic OligoPeptide 10Cys His Phe Pro Phe
Leu Phe Glu Gly Thr Ser Tyr Ser Ser Cys1 5 10 151115PRTArtificial
SequencePUFFERFISH Synthetic OligoPeptide 11Cys His Phe Pro Phe Arg
Phe Gln Asn Lys Pro Tyr Lys His Cys1 5 10 151215PRTArtificial
SequenceFLOUNDER Synthetic OligoPeptide 12Cys His Phe Pro Phe Thr
Phe Glu Gly Lys Ser Tyr Thr Ser Cys1 5 10 151315PRTArtificial
SequenceBOVINE Synthetic OligoPeptide 13Asp Gln Asp Lys Leu Tyr Gly
Phe Cys Pro Thr Arg Val Asp Ala1 5 10 151415PRTArtificial
SequenceRAT Synthetic OligoPeptide 14Asp Lys Ala Asp Gly Phe Cys
Pro Thr Arg Ala Asp Val Thr Val1 5 10 151515PRTArtificial
SequenceRAT Synthetic OligoPeptide 15Asp Gln Asp Lys Leu Tyr Gly
Phe Cys Pro Thr Arg Val Asp Ala1 5 10 151615PRTArtificial
SequenceCARP Synthetic OligoPeptide 16Asp Lys Lys Tyr Gly Phe Cys
Pro Asn Arg Asp Thr Ala Val Ile1 5 10 151715PRTArtificial
SequenceRABBIT Synthetic OligoPeptide 17Asp Lys Asp Lys Leu Tyr Gly
Phe Cys Pro Thr Arg Ala Asp Ser1 5 10 15
* * * * *