U.S. patent application number 15/619559 was filed with the patent office on 2017-11-23 for core-shell particle formulation for delivering multiple therapeutic agents.
This patent application is currently assigned to Amrita Vishwa Vidyapeetham. The applicant listed for this patent is MANZOOR KOYAKUTTY, SHANTIKUMAR NAIR, ARCHANA P.RETNAKUMARI. Invention is credited to MANZOOR KOYAKUTTY, SHANTIKUMAR NAIR, ARCHANA P.RETNAKUMARI.
Application Number | 20170333365 15/619559 |
Document ID | / |
Family ID | 53701643 |
Filed Date | 2017-11-23 |
United States Patent
Application |
20170333365 |
Kind Code |
A1 |
KOYAKUTTY; MANZOOR ; et
al. |
November 23, 2017 |
CORE-SHELL PARTICLE FORMULATION FOR DELIVERING MULTIPLE THERAPEUTIC
AGENTS
Abstract
A core-shell particle formulation for delivering multiple
therapeutic agents is disclosed. More particularly, the formulation
comprising protein-protein core-shell particle configured to
independently release therapeutic agents from the core and the
shell. Moreover, the core-shell particle bearing therapeutic agents
enable treatment against the diseases such as cancer, inflammatory
and auto-immune diseases.
Inventors: |
KOYAKUTTY; MANZOOR; (Kochi,
IN) ; NAIR; SHANTIKUMAR; (Kochi, IN) ;
P.RETNAKUMARI; ARCHANA; (Kochi, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KOYAKUTTY; MANZOOR
NAIR; SHANTIKUMAR
P.RETNAKUMARI; ARCHANA |
Kochi
Kochi
Kochi |
|
IN
IN
IN |
|
|
Assignee: |
Amrita Vishwa Vidyapeetham
Kochi
IN
|
Family ID: |
53701643 |
Appl. No.: |
15/619559 |
Filed: |
June 12, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14585013 |
Dec 29, 2014 |
9707186 |
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15619559 |
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PCT/IN2013/000141 |
Mar 12, 2013 |
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14585013 |
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14465521 |
Aug 21, 2014 |
9402918 |
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PCT/IN2013/000141 |
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PCT/IN2013/000108 |
Feb 19, 2013 |
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14465521 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 31/44 20130101; A61K 47/645
20170801; A61K 31/704 20130101; A61K 47/643 20170801; A61K 31/713
20130101; A61K 39/44 20130101; A61K 9/51 20130101; A61K 47/6851
20170801; A61K 38/00 20130101; A61K 9/167 20130101; A61K 47/644
20170801; A61K 9/0019 20130101; A61K 31/436 20130101; A61K 31/506
20130101; A61K 31/44 20130101; A61K 38/40 20130101; A61K 31/713
20130101; A61K 9/5169 20130101; A61K 47/6803 20170801; A61K 31/704
20130101; A61K 47/6929 20170801; A61K 31/436 20130101; A61K 31/506
20130101; A61K 47/6811 20170801; A61K 45/06 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 31/44 20060101 A61K031/44; A61K 47/68 20060101
A61K047/68; A61K 9/00 20060101 A61K009/00; A61K 31/506 20060101
A61K031/506; A61K 31/436 20060101 A61K031/436; A61K 45/06 20060101
A61K045/06; A61K 31/713 20060101 A61K031/713 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 21, 2012 |
IN |
644/CHE/2012 |
Jun 27, 2012 |
IN |
2550/CHE/2012 |
Claims
1. A core-shell particle formulation for delivering multiple
therapeutic agents comprising: a protein core comprising a first
protein loaded with a first therapeutic agent; and a protein shell
encapsulating the core to form a particle formulation, wherein the
protein shell comprises a cross-linked second protein loaded with a
second therapeutic agent; wherein the particle is configured to
independently release the therapeutic agents from the core and the
shell.
2. The formulation of claim 1, wherein the size of the core-shell
particle is 1-1000 nm.
3. The formulation of claim 1, wherein the first protein or the
second protein comprises human serum albumin, bovine serum albumin,
protamine, transferrin, lactoferrin, fibrinogen, gelatin, mucin,
soy protein, apoferritin, ferritin, lectin, gluten, whey protein,
prolamines, gliadin, hordein, secalin, zein, avenin, or
combinations thereof.
4. The formulation of claim 1, wherein the first therapeutic agent
or the second therapeutic agent comprises cytotoxic drugs, small
molecule kinase inhibitors, phytochemicals, deoxyribozymes,
ribozymes, siRNA, shRNA, DNA, PNAs, miRNAs, or combinations
thereof.
5. The formulation of claim 1, wherein the first therapeutic agent
or the second therapeutic agent comprises demethylation agents,
retinoids, antimetabolites, anti microtubule agents,
anti-angiogenesis agents, alkylating agents, biologic response
modifiers, antitumor antibiotics, proteasome inhibitors,
topoisomerase I inhibitors, hormones, immunomodulators, monoclonal
antibodies, aromatase inhibitors, glucocorticosteroids, cytokines,
enzymes, anti-androgen molecules, epigenetic modifiers, imatinib,
sorafenib, nilotinib, erlotinib, gefitinib, dasatinib, everolimus,
or combinations thereof.
6. The formulation of claim 1, wherein the core or the shell or
both are embedded with metallic nanoclusters comprising one or more
of gold, silver, platinum, copper, or iron.
7. The formulation of claim 1, wherein the therapeutic agents are
configured to be delivered by passive targeting.
8. The formulation of claim 7, wherein the therapeutic agents are
configured to be delivered from the shell and core
sequentially.
9. The formulation of claim 7, wherein the therapeutic agents are
configured to be delivered from the shell and core
simultaneously.
10. The formulation of claim 1, wherein the therapeutic agents are
configured to be delivered by active targeting.
11. The formulation of claim 8, wherein the therapeutic agents are
configured to be delivered from the shell and core
sequentially.
12. The formulation of claim 8, wherein the therapeutic agents are
configured to be delivered from the shell and core
simultaneously.
13. The formulation of claim 8, wherein the active targeting is
achieved by conjugating the core-shell formulation with monoclonal
antibody against CD20, CD33, CD34, CD38, CD44, CD47, CD52 CD90, CD
123, CD 133, EGFR, PDGFR, VEGF, HER2, mTOR, PI3K-Akt, BCR-ABL, SRC,
STAT5, MAPK, HER2, transferrin receptors, R.GD, CRGD, LyP-1,
bombesin, FSH33, truncated human basic fibroblast growth factor,
octreotide, folic acid, mannose, hyaluronic acid, transferrin,
somatostatin, or aptamers.
14. A method of treating a disease, condition or disorder
comprising: administering to a human patient a therapeutically
effective amount of a core-shell particle formulation comprising a
protein core comprising a first protein loaded with a first
therapeutic agent; and a protein shell encapsulating the core to
form a particle formulation, wherein the protein shell comprises a
cross-linked second protein loaded with a second therapeutic agent;
wherein the particle is configured to independently release the
therapeutic agents from the core and the shell; wherein the
core-shell particle formulation is administered by local injection,
intravenous, subcutaneous, intramuscular or oral delivery.
15. The method of claim 14, further comprising passively targeting
a targeted tissue with the therapeutic agents.
16. The method of claim 14, further comprising actively targeting a
targeted tissue with the therapeutic agents.
17. The method of claim 16, further comprising sequentially
delivering the therapeutic agents from the shell and core to the
targeted tissue.
18. The method of claim 16, further comprising simultaneously
delivering the therapeutic agents from the shell and core to the
targeted tissue.
19. The method of claim 14, wherein the treatment comprises
anticancer therapy, anti-inflammatory therapy, or immunotherapy.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/585,013 filed on Dec. 29, 2014, which is a
continuation-in-part of PCT international application No.
PCT/IN2013/000141 filed on Mar. 12, 2013, which claims priority to
Indian patent application No. 2550/CHE/2012, filed on Jun. 27,
2012, and a continuation-in-part of U.S. application Ser. No.
14/465,521 filed on Aug. 21, 2014, which is a continuation of PCT
international application No. PCT/IN2013/00108 filed on 19 Feb.
2013, which claims priority to Indian patent application No.
644/CHE/2012, filed on 21 Feb. 2012, the full disclosures of which
are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to core-shell particle
formulations for delivering multiple therapeutic agents, and more
particularly, core-shell particle formulation configured to
independently release therapeutic agents from the core and the
shell. The core-shell particle bearing therapeutic agents are
envisaged to enable treatment against diseases such as cancer,
inflammatory and auto-immune diseases.
DESCRIPTION OF THE RELATED ART
[0003] Successful management of diseases requires development of
drug delivery systems with maximum therapeutic benefits. Most of
the diseases including cancer are associated with deregulation of
multiple signaling pathways. An essential requirement of drug
delivery systems is the controlled delivery of a therapeutic
molecule to the diseased site therapeutically relevant
concentrations. The site-specific delivery of multiple therapeutic
molecules to the diseased site using a single carrier vehicle in a
specified steady concentration for prescribed time duration
improves the efficacy of the therapeutic molecule and thus reduces
the possible side effects, thus improving the therapeutic index.
The release kinetics of the therapeutic molecule is often dependent
upon the encapsulating material/carrier properties, drug-particle
interactions or through some other trigger mechanisms, which assist
in the drug release. Design of drug delivery systems generally
involves encapsulation of the drug within a suitable shell to form
particles of suitable size. The drug can be distributed either
within a hollow shell or within the solid particle.
[0004] The advantages of such encapsulation is the control over
release kinetics, giving the ability for slow release over a long
period of time, and protection of the drug from a potentially
degrading biological environment. Recent advancements in
nanotechnology have revolutionized the field of drug delivery. The
advantages of nanoparticles over conventional systems of drug
delivery include, high surface area to volume ratio enabling better
cellular uptake, thereby affecting intracellular pathways of action
compared to that of free molecules and the ability to efficiently
bio-functionalize the particulate surface with cell-specific
targeting ligands for specific attachment to particular cells which
require drug action. Protein based drug delivery systems are ideal
platforms for the delivery of multiple therapeutics for in vivo
applications due to their amphiphilic nature, biocompatibility and
biodegradability coupled with low toxicity. The degradation
products of the carrier system will be amino acids, which are well
tolerated by the human body.
[0005] Depending upon the nature of the molecules to be
encapsulated, a wide choice of preparations is available such as
desolvation, heat denaturation, coacervation, cross-linking, nano
precipitation emulsification, etc. The particle size of the system
can be fine-tuned with slight changes in synthesis parameters such
as temperature, pH, etc. Moreover, the nanoparticles possess
greater stability during storage or in vivo after administration,
and provide surface functional groups for conjugation to cancer
targeting ligands. They also are suitable for administration
through different routes.
[0006] US20101122077 describes combination therapy methods of
treating proliferative diseases like cancer with a first therapy
comprising of effective amount of a taxane in a nanoparticle
composition, with second therapy such as radiation, surgery,
administration of chemotherapeutic agents such as anti-VEGF
antibody or combinations thereof
[0007] Most of the FDA approved nanoformulations and other drug
delivery systems reported till date are single agent delivery
vehicles which pose structural constraints in encapsulation and
release of multiple payloads in optimal concentrations at the tumor
site. Encapsulation of more than one drug in the same nano-carrier
may elicit undesirable drug-drug interaction which might alter the
pharmacology of both the drugs, resulting in inefficacy of the
drugs. The conventional chemotherapy regimen in an attempt to
reduce the tumor volume, do not discriminate between rapidly
dividing normal cells and tumor cells, thus leading o severe
side-effects.
[0008] Therefore, there remains a need for a drug delivery system
for delivering combination therapies so that each agent provides
the desired maximal effect. Moreover, the drug delivery system must
deliver multiple therapeutic agents and independently release these
therapeutic agents to the targeted diseased sites.
SUMMARY OF THE INVENTION
[0009] A core-shell particle formulation for delivering multiple
therapeutic agents is disclosed. The formulation comprises one or
more proteins forming a core; and one or more proteins forming a
shell encapsulating the core to form a particle formulation. The
core and the shell each comprise one or more therapeutic agents,
and the particle is configured to independently release therapeutic
agents from the core and the shell. In some embodiments the the
size of the core-shell particle is 1-1000 nm.
[0010] The proteins in the formulation are chosen from the group
consisting of human serum albumin, bovine serum albumin, protamine,
transferrin, lactoferrin, fibrinogen, gelatin, mucin, soy protein,
apoferritin, ferritin, lectin, gluten, whey protein, prolamines,
gliadin, hordein, secalin, zein, avenin, and combinations thereof.
In some embodiments of the formulation, the core and shell are
loaded with different therapeutic agents from the group consisting
of cytotoxic drugs, small molecule kinase inhibitors,
phytochemicals, deoxyribozymes, ribozymes, siRNA, shRNA, DNA, PNAs,
miRNAs, and combinations thereof.
[0011] In some embodiments of the formulation the therapeutic
agents are chosen from the group consisting of demethylation
agents, retinoids, antimetabolites, antimicrotubule agents,
anti-angiogenesis agents, alkylating agents, biologic response
modifiers, antitumor antibiotics, proteasome inhibitors,
topoisomerase I inhibitors, hormones, immunomodulators, monoclonal
antibodies, aromatase inhibitors, glucocorticosteroids, cytokines,
enzymes, anti-androgen molecules, epigenetic modifiers, imatinib,
sorafenib, nilotinib, erlotinib, gefitinib, dasatinib, everolimus,
and combinations thereof. In some embodiments the core or the shell
or both are embedded with metallic nanoclusters comprising one or
more of gold, silver, platinum, copper, or iron.
[0012] In some embodiments the therapeutic agents are configured to
be delivered by passive targeting from the shell and core either
sequentially or simultaneously. In other embodiments the
therapeutic agents are configured to be delivered by active
targeting from the shell and core either sequentially or
simultaneously. The formulation delivered by active targeting is
achieved by conjugating the core-shell formulation with monoclonal
antibody against CD20, CD33, CD34, CD38, CD44, CD47, CD52 CD90, CD
123, CD 133, EGFR, PDGFR, VEGF, HER2, mTOR, PI3K-Akt, BCR-ABL, SRC,
STAT5, MAPK, HER2, transferrin receptors, R.GD, CRGD, LyP-1,
bombesin, FSH33, truncated human basic fibroblast growth factor,
octreotide, folic acid, mannose, hyaluronic acid, transferrin,
somatostatin, or aptamers.
[0013] A method of treating a disease, condition or disorder is
disclosed, comprising administering to a human patient a
therapeutically effective amount of a core-shell particle
formulation comprising one or more proteins forming a core and one
or more proteins forming a shell encapsulating the core to form a
particle formulation; wherein the core and the shell each comprise
one or more therapeutic agents, and wherein the particle is
configured to independently release therapeutic agents from the
core and the shell. The core-shell particle formulation may be
administered by local injection, intravenous, subcutaneous,
intramuscular or oral delivery.
[0014] The method may employ either passively targeting a targeted
tissue with the therapeutic agents, or the targeting may be active,
and the agents may be delivered from the shell and core either
sequentially or simultaneously. The treatment may comprise
anticancer therapy, anti-inflammatory therapy, or
immunotherapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The invention has other advantages and features which will
be more readily apparent from the following detailed description of
the invention and the appended claims, when taken in conjunction
with the accompanying drawings, in which:
[0016] FIG. 1 illustrates a nanoparticle core-shell formulation
according to one embodiment.
[0017] FIG. 2 is a schematic of the method of preparing a
core-shell formulation of the invention according to one
embodiment.
[0018] FIGS. 3A and 3B show an example with two rapamycin and
dasatinib docked protamine and albumin respectively, in a
core-shell architecture, FIG. 3A shows computational modeling of
albumin-dasatinib and protamine-rapamycin interactions, FIG. 3B
shows computationally designed structure of (protamine-rapamycin)
(albumin-dasatinib) core-shell nanoparticles.
[0019] FIGS. 4A-C illustrate the size distribution of
(protamine-rapamycin) and (albumin-dasatinib) core-shell
system.
[0020] FIG. 5A and FIG. 5B show the photoluminescence spectra of
(protamine-rapamycin) and (albumin-dasatinib) core-shell system,
where albumin is doped with metallic nanoclusters of gold.
[0021] FIG. 6A show the red-NIR emission from gold nanocluster
doped protein-protein core-shell nanomedicine and FIG. 6B show the
corresponding photoluminescence excitation-emission spectrum.
[0022] FIG. 7 is illustrates the improved efficacy of the
(prt-rapa)-(alb-dasa) core-shell nanomedicine in cancer cell
migratory potential. The encircled image shows the distorted
morphology of breast adenocarcinoma cells treated with the
protein-protein core-shell nanomedicine comprising of gold
nanocluster doped (prt-rapa)-(alb-dasa).
[0023] FIGS. 8A-C show the destabilization of cytoskeleton and
distortion of cellular morphology by (protamine-rapamycin) and
(albumin-dasatinib) core-shell system as depicted by actin
staining.
[0024] FIGS. 9A-C show the quantitative data with substantially
improved cytotoxicity of (prt-rapa)-(tf-dasa) core-shell system in
highly aggressive breast carcinoma cells with increasing
concentrations of rapamycin and constant dasatinib
concentration.
[0025] FIG. 10 shows UV-VIS absorption spectra of
(protamine-imatinib) and (transferrin-sorafenib) core-shell
nanomedicine.
[0026] FIG. 11 is a scanning electron microscopic image of
(protamine-imatinib) and (transferrin-sorafenib) core-shell
nanomedicine showing spherical particles of 200 nm.
[0027] FIG. 12 illustrates the improved cytotoxicity of
(protamine-imatinib) and (transferrin-sorafenib) core-shell
nanomedicine in drug resistant CML cells carrying amplification of
BCR-ABL and STAT5 activation.
[0028] FIGS. 13A-D illustrate the size distribution and
morphological characterization of
(protamine-siRNA)-(transferrin-soraf) core-shell nanoparticles
using OLS AFM (FIG. 13A and 13B) and SEM (FIG. 13C and 13D) showing
PS-siRNA nanocore of .about.20 nm and
(protamine-siRNA)-(transferrin-soraf) core-shell nanoparticles of
size .about.200 nm.
[0029] FIG. 14A shows cytotoxic effect of
(protamine-siRNA)-(transferrin-soraf) in drug resistant CML cells
carrying amplification of BCR-ABL and STAT5 activation and FIG. 14B
shows normal healthy cells, which remain 100% viable upon treatment
with (protamine-siRNA) and (transferrin-soraf).
[0030] FIGS. 15A-F demonstrate genomic level effect of the
core-shell nanoparticles, FIG. 15A shows dose dependent
cytotoxicity in K562 CML cells by protamine-siRNA nano-core, FIG.
15B shows silencing of BCR-ABL oncogene, confirmed by immunoblot
(FIG. 15C). FIG. 15D-F show cytotoxicity of transferrin-sorafenib
nanoshell through inhibition of phosphor-STAT5.
[0031] FIGS. 16A-N show cytotoxicity of protamine siRNA-Albumin
Sorafenib nanomedicine in vitro, on leukemic cells from 14 patients
exhibiting enhanced cytotoxicity against core-shell NPs compared to
core alone or shell alone.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] While the invention has been disclosed with reference to
certain embodiments, it will be understood by those skilled in the
art that various changes may be made and equivalents may be
substituted without departing from the scope of the invention. In
addition, many modifications may be made to adapt to a particular
situation or material to the teachings of the invention without
departing from its scope.
[0033] Throughout the specification and claims, the following terms
take the meanings explicitly associated herein unless the context
clearly dictates otherwise. The meaning of "a", "an", and "the"
include plural references. The meaning of "in" includes "in" and
"on." Referring to the drawings, like numbers indicate like parts
throughout the views. Additionally, a reference to the singular
includes a reference to the plural unless otherwise stated or
inconsistent with the disclosure herein.
[0034] The term "nanomedicine" as used herein may refer to
nanoparticles of two or more proteins, measuring size about 1-1000
nm capable of delivering multiple anti-cancer agents such as
chemotherapeutic drugs, small molecule inhibitors etc., in
different combinations of at least one small molecule kinase
inhibitor and one chemotherapeutic drug or suitable combination of
two small molecule inhibitors/chemotherapeutic drugs together. In
one embodiment the nanoparticles have a size around 1-500 nm. In
another embodiment the nanoparticles have a size around 1-200 nm in
size.
[0035] "Protein-protein core-shell nanomedicine" may refer to
nanomedicine constructs comprising a nano-core formed by one type
of protein loaded with one type of chemotherapeutic drug and an
outer nano-shell formed by another type of protein loaded with
another drug.
[0036] Therapeutics may be refer to synthetic drugs including
cytotoxic drugs and small molecule kinase inhibitors,
phytochemicals or nucleic acid drugs such as siRNAs, shRNAs,
miRNAs, PNAs, DNA, DNAzymes, ribozymes, or prodrugs thereof, that
have a therapeutic effect against diseases including cancer,
inflammatory and auto-immune diseases and the like.
[0037] The proposed invention relating to core-shell particle
formulation for delivering multiple therapeutic agents is described
in the following sections referring to the sequentially numbered
figures. The above-mentioned objectives are achieved through the
core-shell particle bearing therapeutic agents specifically
targeted to the preferred site of action and configured to
controllably release therapeutic agents.
[0038] In one embodiment, core-shell particles for delivering
multiple therapeutic agents and methods for their preparation are
disclosed, as shown in FIGS. 1 and 2, respectively. As shown in
FIG. 1, in one embodiment, the particles of the invention comprise
one or more proteins to form a core 101 and one or more proteins
forming a shell 106. In various embodiments, the core 101 and the
shell 106 each comprise one or more therapeutic agents. In one
embodiment of the invention illustrated in FIG. 2, the particle is
obtained using the steps shown in the figure. In step 201, a
protein precursor solution of the core 101 is reacted with the
first therapeutic agent 102 and precipitated to form the
drug-loaded core nanoparticles 103 in step 202. In step 203, a
second therapeutic agent 104 is blended with a second protein
solution 105 for forming the protein shell. The drug-loaded core
nanoparticles 103 are added to the blended second protein solution
105 in step 204, in which the therapeutic agent 104 is incorporated
into the protein 105 and crosslinked to form a shell 106 around the
core 103. Finally, in step 205, the fully formed core-shell
nanoparticles 110 with the first therapeutic agent 102 loaded in
the core and the second therapeutic agent 104 loaded in the shell
are separated from solution for therapeutic use.
[0039] In various embodiments, the protein 105 forming the core 101
and shell 106 is chosen from human serum albumin, bovine serum
albumin, protamine, transferrin, lactoferrin, fibrinogen, gelatin,
mucin, soy protein, apoferritin, ferritin, lectin, gluten, whey
protein, prolamines such as gliadin, hordein, secalin, zein,
avenin, or combinations thereof.
[0040] In various embodiments the protein nanoparticle or the
protein shell is formed by a method that is one of
nano-precipitation, coacervation, self-assembly, cross-linking,
spray drying, electrospray, emulsion desolvation, snap injection,
etc. In some embodiments, one or both of the proteins in the
core-shell nanoparticle 110 are doped/loaded/embedded with metallic
nanoclusters comprising one or more of gold, silver, platinum,
copper, or iron for the purpose of tracking the nanoparticles in
vivo using one or more of optical, magnetic or x-ray contrast. In
some embodiments, the prepared core-shell nanoparticle 110 is
purified by centrifugation and lyophilisation.
[0041] In one embodiment, the total size of the core-shell particle
is of 1-1000 nm. In various embodiments, the core and shell are
loaded with one or more small molecule kinase inhibitors or
chemotherapeutic drugs. In various embodiments, the shell comprises
either hydrophilic or hydrophobic therapeutic agents, or both types
of agents.
[0042] In one embodiment, the core and shell are loaded with
different therapeutic agents comprising synthetic chemotherapeutic
drugs including cytotoxic drugs or one or more small molecule
kinase inhibitors or phytochemicals or nucleic acid drugs such as
deoxyribozymes, ribozymes, siRNA, shRNA, DNA, PNAs, or miRNAs or
combinations thereof.
[0043] In various embodiments, the chemotherapeutic drug is chosen
from one or more of demethylation agents, retinoids,
antimetabolites, antimicrotubule agents, anti-angiogenesis agents,
alkylating agents, biological response modifiers, antitumor
antibiotics, proteasome inhibitors, topoisomerase I inhibitors,
hormones, immunomodulators, monoclonal antibodies, aromatase
inhibitors, glucocorticosteroids, cytokines, enzymes, anti-androgen
molecules, epigenetic modifiers, or small molecule kinase
inhibitors including imatinib, sorafenib, nilotinib, erlotinib,
gefitinib, dasatinib, everolimus.
[0044] In various embodiments, the nanoparticles comprising the
therapeutic agents 102 and 104 are configured to be delivered as
formulations with excipients suitable for local injection, or
intravenous, subcutaneous, intramuscular or oral delivery. In some
embodiments the formulations are configured to deliver therapeutic
agents to targeted tissue by either passive or active targeting. In
one embodiment, the active targeting is done by conjugating the
core-shell formulation with targeting ligands such as monoclonal
antibody against receptors such as, CD20, CD33, CD34, CD38, CD44,
CD47, CD52 CD90, CD 123, CD 133, EGFR, PDGFR, VEGF, HER2, mTOR,
PI3K-Akt, BCR-ABL, SRC, STAT5, MAPK, HER2, transferrin receptors
and like, peptides such as R.GD, CRGD, LyP-1, bombesin (BBN),
FSH33, truncated human basic fibroblast growth factor (tbFGF),
octreotide, folic acid, mannose, hyaluronic acid (HA), proteins
such as transferrin, somatostatin or aptamers. In one embodiment,
the therapeutic agents are configured to be delivered to tissue
from the shell and core either sequentially or simultaneously.
[0045] In various embodiments, a method of treatment of treatment
against cancer, inflammatory or auto-immune diseases is disclosed,
comprising delivering to targeted tissue, a therapeutically
effective amount of a formulation comprising the core-shell
nanoparticles as illustrated in various earlier embodiments. The
method in various embodiments may involve administering the
formulation to a human patient by local injection, intravenous,
subcutaneous, intramuscular or oral delivery. The method in some
embodiments may target the therapeutic agents to specific
tissue.
[0046] The method in some embodiments may involve either
simultaneous release of the therapeutic agents from the core of the
nanoparticles, and in other embodiments the agents may be released
sequentially, as required. In some embodiments, the method may use
passive targeting of tissue, while in other embodiments the
therapeutic agents are delivered by active targeting. In some
embodiments of the method the nanoparticles are tracked in vivo
using one or more of optical, magnetic or x-ray contrast.
[0047] The particle formulations disclosed in the various
embodiments above are configured to independently release first
therapeutic agents 102 from the core 101 and second therapeutic
agents 104 from the shell 106. This gives the nanocarrier an
extraordinary ability to deliver multiple therapeutic molecules
directly to specific cells rather than systemically to all cells
and, further, to deliver both the drugs into the cell, thereby
potentially reducing dosages at equivalent efficacy. Further the
use of two separate protein phases is envisioned, rather than
synthetic materials as in engineered nanoparticles. Use of proteins
as a nanocarrier is considered because of the reduced toxicity of
such natural materials. A second part of this invention is the
modification of one or more of the proteins by doping/embedding
them with metallic nanoclusters of gold or other suitable metals
nanoclusters for imparting specific characteristic properties to
the nanocarrier such as optical/magnetic/x-ray contrast. This
enables tracking of the nanoparticles in-vivo and understanding
their bio-distribution and location.
[0048] One application wherein such a drug delivery system can be
especially useful is in cancer or tumor formation. Tumorigenesis is
a multi-step process, where the genetic alterations enable the
cancer cells to acquire properties such as self-sufficiency of
growth signals, insensitivity to anti-growth signals evasion of
apoptosis, limitless replicative potential, sustained angiogenesis
which further lead to tissue invasion and metastasis. Unlike the
cytotoxic chemotherapeutic drugs, protein kinase inhibitors target
specifically the protein kinases, which are deregulated
(constitutively activated/mutated/over-expressed) in cancer cells.
Moreover, most of the kinase inhibitors have been found to have low
levels of undesirable side effects in clinical and preclinical
studies compared to cytotoxic drugs. Yet, for highly aggressive and
metastatic cancers, cytotoxic drugs present an immediate effect
compared to kinase inhibitors. Resistance to kinase inhibitors in
the long run due to point mutations in the drug-binding domain of
kinases eventually distorts the conformation of drug binding domain
and hence prevents the drug from binding to it. In certain cases,
kinase inhibition of the primary oncoprotein can lead to the
activation or over-expression of a secondary survival signal in the
oncogenic network. In those cases monotherapy with a single
therapeutic agent remains ineffective. Combination of cytotoxic
chemotherapeutic drugs and kinase inhibitors can be an attractive
approach to treat highly aggressive tumor masses. The current
invention enables combination therapy using bio-friendly protein
based nanocarriers, and attempts to solve most of the issues
associated with conventional treatment strategy.
[0049] The invention is further illustrated with reference to the
following examples, which however, are not to be construed to limit
the scope of the invention as defined by the appended claims.
EXAMPLES
Example--1
Synthesis of (Protamine-Rapamycin) Nanocore:
[0050] Synthesis of protamine-rapamycin nanocore:
protamine-rapamycin nanocore was prepared using aqueous chemical
route. The cationic peptide protamine (10 kDa) was dissolved at a
concentration of 11 mg/ml in nuclease and endotoxin free water.
Rapamycin was dissolved in DMSO as per the manufacturer's
instructions. Rapamycin was added to aqueous solution of protamine.
The complexation of protamine and rapamycin resulted in a turbid
solution, which was vortexed vigorously and incubated at room
temperature for 30 min to enable the effective complexation of
rapamycin with protamine. Thus formed protamine-rapamycin nanocore
was purified by dialysis using 2 kDa cut off dialysis membrane.
Synthesis of (Protamine-Rapamycin)-(Albumin-Dasatinib) Core-Shell
Nanomedicine:
[0051] Dasatinib in DMSO was mixed with aqueous solution of albumin
at a dasatinib final concentration of 100 .mu.M. In a typical
synthesis, 100 .mu.M dasatinib in albumin was added drop-wise to
protamine-rapamycin solution. The solution was kept under
continuous stirring for .about.30 m at room temperature. The
individual HSA molecules were crosslinked using 2 mg
1-Hhyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC), a
zero-length cross-linker for effective entrapment of the
therapeutic molecules m the protein shell. The reaction was
continued for--2 h at room temperature. The solution was further
subjected to dialysis using dialysis cassettes with 2 kDa molecular
weight cut off and lyophilized for 48 h. The percentage entrapment
of the drug was determined from standard graph of rapamycin and
dasatinib.
Example--2
Computational Modeling of (Protamine-Rapamycin)-(Albumin-Dasatinib)
Core-Shell Nanomedicine:
[0052] The computational modeling of two different drug molecules
(rapamycin and dasatinib) docked to two different proteins
(protamine and albumin) in a core-shell architecture is designed.
Computational modeling of albumin-dasatinib and protamine-rapamycin
interactions is shown in FIG. 3A and computationally designed
structure of (protamine-rapamycin) (albumin-dasatinib) core-shell
nanoparticles is shown in FIG. 3B.
Characteristics of (Protamine-Rapamycin)-(Albumin-Dasatinib)
Core-Shell Nanomedicine:
[0053] FIG. 4 shows an example of the result with the size
distribution of (protamine-rapamycin) and (albumin-dasatinib) (FIG.
4A) core-shell system using dynamic light scattering (DLS) showing
a core of size around 45 nm and a core-shell structure of size
around 158 nm (FIG. 4B) atomic force microscopy (AFM) and (FIG. 4C)
scanning electron microscopy showing the spherical morphology of
the particles formed. The photoluminescence spectra of
(protamine-rapamycin) and (albumin-dasatinib) core-shell system,
where albumin is doped with metallic nanoclusters of gold is shown
in FIG. 5A and 5B. Further, the red-NIR emission from gold
nanocluster doped protein-protein core-shell nanomedicine and the
corresponding photoluminescence excitation-emission spectrum is
shown in FIG. 6A and 6B.
Example--3
Results of Nanomedicine Treatment Against Breast Cancer:
[0054] The protamine-rapamycin) and (albumin-dasatinib) core-shell
nanomedicine is administered either intravenously, orally,
parenterally, subcutaneously or by direct local delivery. The
method of treatment of cancer-like diseases using core-shell
nanomedicine aiding combinatorial anti-cancer therapy by sequential
or simultaneous delivery of a combination of small molecule kinase
inhibitor and chemodrugs. of the (prt-rapa)-(alb-dasa) core-shell
nanomedicine in cancer cell migratory potential shows an improved
efficacy in FIG. 7. The encircled image shows the distorted
morphology of breast adenocarcinoma cells treated with the
protein-protein core-shell nanomedicine comprising of gold
nanocluster doped (prt-rapa)-(alb-dasa). FIG. 8 shows the
destabilization of cytoskeleton and distortion of cellular
morphology by (protamine-rapamycin) and (albumin-dasatinib)
core-shell system as depicted by actin staining.
[0055] Further in another combination, the treatment shows the
quantitative data with substantially improved cytotoxicity of
(protamine-rapamycin) and (transferrin-dasatinib) core-shell system
is shown in FIG. 9. The improved toxicity against highly aggressive
breast carcinoma cells with increasing concentrations of rapamycin
and constant dasatinib concentration is proved in FIG. 9A, 9B and
9C.
Example--4
Synthesis of Protamine Imatinib-Albumin Sorafenib Nanomedicine
Against Drug Resistant Chronic Myeloid Leukemia (CML)--Synthesis of
Protamine-Imatinib Nanocore:
[0056] Protamine-rapamycin nanocore was prepared using aqueous wet
chemical route. The cationic peptide protamine (10 kDa) was
dissolved at a concentration of 11 mg/ml in nuclease and endotoxin
free water. Imatinib was dissolved m DMSO, as per the
manufacturer's instructions imatinib was added to aqueous solution
of protamine. The solution was vortexed vigorously and incubated at
room temperature for 30 m to enable the effective complexation of
imatinib with protamine.
Characteristics of (Protamine-Imatinib)-(Transferrin-Sorafenib)
Core-Shell Nanomedicine:
[0057] In another combination, the synthesis of
(protamine-imatinib) and (transferrin-sorafenib) core-shell
nanomedicine is done as steps followed in the above method. The
UV-VIS absorption studies are shown in FIG. 10. The scanning
electron microscopic image of (protamine-imatinib) and
(transferrin-sorafenib) core-shell nanomedicine with spherical
particles of .about.200 nm is shown in FIG. 11.
Example--5
Results of (Protamine-Imatinib)-(Transferrin-Sorafenib) Core-Shell
Nanomedicine Against Chronic Myeloid Leukemia (CML):
[0058] The composition of sorafenib with 1 .mu.M in the protamine
shell and imatinib with 5 .mu.M in the transferrin shell shown an
improved cytotoxicity against the chronic myeloid leukemia (CML)
cells. The improved cytotoxicity of (protamine-imatinib) and
(transferrin-sorafenib) core-shell nanomedicine in drug resistant
CML cells carrying amplification of BCR-ABL and STAT5 activation is
shown in FIG. 12.
Example--6
Synthesis of (Protamine-Imatinib)-(Albumin-Dasatinib) Core-Shell
Nanomedicine:
[0059] Sorafenib in DMSO was mixed with aqueous solution of albumin
at a sorafenib final concentration of 100 .mu.M. In a typical
synthesis, 100 .mu.M sorafenib in albumin was added drop-wise to
protamine-imatinib solution. The solution was kept under continuous
stirring, for 30 m at room temperature. The individual HSA
molecules were crosslinked using 2 mg
1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC),
a zero-length cross-linker for effective entrapment of the drug in
the protein shell. The reaction was continued for 2 h at room
temperature. The solution was further subjected to dialysis using
dialysis cassettes with 2 kDa molecular weight cut off and
lyophilized for 48 h. The percentage entrapment of the drug was
determined from standard graph of imatinib and sorafenib.
Example--7
[0060] Synthesis of Protamine siRNA-Albumin Sorafenib Nanomedicine
Conjugated to Transferrin--Synthesis of Protamine siRNA
Nanoconjugates:
[0061] Protamine-siRNA nanoconjugates were prepared using aqueous
wet chemical route, with an N/P ratio of 12. The cationic peptide,
protamine (10 kDa) was dissolved at a concentration of 11 mg/ml in
nuclease and endotoxin free water. Lyophilized powder of siRNA
targeted to BCR-ABL fusion kinase transcript in chronic myeloid
leukemia (CML) was dissolved in RNase free water as per the
manufacturer's instructions to prepare a stock solution of 350 nM.
Protamine solution was added drop-wise to siRNA solution at the
respective N/P ratio. The complexation of protamine and siRNA
resulted in a turbid solution which was vortexed vigorously and
incubated at room temperature for 30 min to enable the effective
complexation of siRNA with protamine.
Characteristics of (Protamine-siRNA)-(Transferrin-Sorafenib)
Core-Shell Nanomedicine.
[0062] The result of the size distribution and morphological
characterization of (protamine-siRNA)-(transferrin-sorafenib)
core-shell nanoparticles using OLS AFM and SEM showing PS-siRNA
nanocore of .about.20 nm and (protamine-siRNA)-(transferrin-soraf)
core-shell nanoparticles of size .about.200 nm is shown in FIG.
13.
Synthesis of Albumin Sorafenib Nanoconjugates:
[0063] Albumin-sorafenib nanoparticles were prepared using an
aqueous wet chemical route. Sorafenib, which is a multi-kinase
inhibitor targeting STAT5 kinase in drug resistant CML was prepared
in DMSO and aliquots were stored at -20.degree. C. In a typical
synthesis, 15.7 mM sorafenib tosylate in DMSO was added drop-wise
to 5 mg/ml HSA solution. The solution was kept under continuous
stirring where the final concentration of sorafenib was adjusted to
500 .mu.M. The stirring was continued for 30 m at room temperature.
The individual HSA molecules were cross-linked using 2 mg
1-Ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EOC),
a zero-length cross-linker for effective entrapment of the drug in
the protein shell. The reaction was continued for .about.2 h at
room temperature. Sorafenib embedded albumin nanoparticles
(nAlb-Soraf) were subjected to dialysis using dialysis cassettes
with 2 kDa molecular weight cut off and lyophilized for 48 h. The
percentage entrapment of the drug was determined from standard
graph of sorafenib prepared in DMSO.
Example--8
[0064] Synthesis of Protamine siRNA-Transferrin Sorafenib
Nanomedicine Doped With Metallic Nanoclusters:
[0065] The preparation of protamine-siRNA nanoconjugates is done as
described in example 3. In this method of preparation, the carrier
protein itself possesses cancer cell specific targeting capability.
Transferrin used for encapsulating the small molecule kinase
inhibitor is pre-doped with metallic nanoclusters of gold,
platinum, silver etc. for imparting specific characteristics such
as optical contrast, magnetic contrast or and/or cationic zeta
potential. The precursors of metals are added to protein solution
at 37.degree. C. at 10 mM concentration kept under stirring. The
reduction of the metal ions to metallic nanoclusters is aided by
reducing agents such as NaOH, ascorbic acid etc. The resulting
solution is purified using desalting columns and further used for
preparing transferrin-sorafenib nanoconjugates as described in
example 3, which is further used for complexation with
protamine-siRNA nanoconjugates to form protein-protein composite
nanomedicine.
Example--9
Results of Nanomedicine Treatment Against Chronic Myeloid Leukemia
(CML):
[0066] The protamine siRNA-albumin sorafenib nanomedicine treated
against immune disease such as chronic myeloid leukemia (CML) shows
an example with the improved result in FIG. 14. The cytotoxic
effect of (protamine-siRNA)-(transferrin-soraf) in drug resistant
CML cells carrying amplification of BCR-ABL and STAT5 activation is
shown in FIG. 14A. The normal healthy cells, which remain 100%
viable upon treatment with (protamine-siRNA) and
(transferrin-soraf) is shown in FIG. 14B.
Example--10
Genomic Level and Proteomic Level Effect of the Core-Shell
Nanoparticles:
[0067] This example, as depicted in FIG. 15A-F shows genomic level
effect of the protamine-siRNA nanocore in silencing the onco-gene,
BCR-ABL, and protein (STAT5) inhibition effect of
transferrin-Sorafenib nano-shell. FIG. 15A shows dose dependent
cytotoxicity in K562 CML cells by nano-core at various
concentrations and FIG. 15B shows the mechanism of toxicity as due
to the silencing of BCR-ABL oncogene, which is further confirmed by
immunoblot (FIG. 15C) where BCR-ABL onco-protein and its downstream
signaling phosphorylated CRKL was found inhibited. On the other
side, nano-shell exerted its enhanced effect of cytotoxicity
compared (FIG. 15D) to free sorafenib through protein level
inhibition of phospho-STAT5 and its downstream pathway Mcl-1 (FIG.
15E and 15F). Clearly, this example demonstrates the independent
molecular level activity of the constituents of the core-shell
nanoparticles.
Example--11
[0068] Effect of Protamine siRNA-Albumin Sorafenib Nanomedicine in
CML Patient Cells:
[0069] In this example we show the clinical significance of the
core-shell nanoparticles by imparting synergistic cytotoxicity in
actual patient samples, derived from n=14 CML patients. For this
testing, we have isolated the blast population of leukemic cells
from the patients at various stages of the disease and treated
these cells in vitro with nano-core alone, nano-shell alone or
nano-core-shells. As seen from the FIG. 16, all patient samples
(P1-P14) showed enhanced cytotoxicity against core-shell NPs
compared to core alone or shell alone. 10 .mu.M concentration of
sorafenib was used in nanoshell and 2 nm or 5 nm of siRNA against
BCR-ABL was used in nanocore.
* * * * *