U.S. patent application number 15/198153 was filed with the patent office on 2017-07-13 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 PARWATHY CHANDRAN, MANZOOR KOYAKUTTY, SHANTIKUMAR NAIR, ARCHANA P.R.. Invention is credited to PARWATHY CHANDRAN, MANZOOR KOYAKUTTY, SHANTIKUMAR NAIR, ARCHANA P.R..
Application Number | 20170196811 15/198153 |
Document ID | / |
Family ID | 48576479 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170196811 |
Kind Code |
A1 |
KOYAKUTTY; MANZOOR ; et
al. |
July 13, 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, core-shell
particle formulation configured to independently release
therapeutic agents from the core and the shell. Moreover, the
core-shell particle bearing therapeutic agents enables treatment
against the diseases such as cancer, inflammatory and auto-immune
diseases.
Inventors: |
KOYAKUTTY; MANZOOR; (Kochi,
IN) ; CHANDRAN; PARWATHY; (Kochi, IN) ; P.R.;
ARCHANA; (Kochi, IN) ; NAIR; SHANTIKUMAR;
(Kochi, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KOYAKUTTY; MANZOOR
CHANDRAN; PARWATHY
P.R.; ARCHANA
NAIR; SHANTIKUMAR |
Kochi
Kochi
Kochi
Kochi |
|
IN
IN
IN
IN |
|
|
Assignee: |
Amrita Vishwa Vidyapeetham
Kochi
IN
|
Family ID: |
48576479 |
Appl. No.: |
15/198153 |
Filed: |
June 30, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14465521 |
Aug 21, 2014 |
9402918 |
|
|
15198153 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5169 20130101;
A61K 9/1647 20130101; A61K 9/1658 20130101; A61K 31/337 20130101;
A61K 31/506 20130101; A61K 9/1635 20130101; A61K 31/436 20130101;
A61K 31/704 20130101; A61K 31/44 20130101; A61K 47/6931 20170801;
A61K 45/06 20130101; A61K 47/6939 20170801; A61K 47/6803 20170801;
A61K 47/6927 20170801; A61K 9/1652 20130101; A61K 47/6933 20170801;
A61K 9/5161 20130101; A61K 9/5192 20130101; A61K 9/5138 20130101;
A61K 9/167 20130101; A61K 9/5153 20130101; A61K 47/6935 20170801;
A61K 31/437 20130101 |
International
Class: |
A61K 9/16 20060101
A61K009/16; A61K 31/704 20060101 A61K031/704; A61K 31/506 20060101
A61K031/506; A61K 31/337 20060101 A61K031/337; A61K 31/436 20060101
A61K031/436; A61K 31/44 20060101 A61K031/44 |
Claims
1. A core-shell particle formulation for delivering multiple
therapeutic agents comprising: one or more polymers forming a core,
wherein the one or more polymers forming the core comprise
poly(lactic-co-glycolic acid); and one or more proteins forming a
shell encapsulating the core to form a particle formulation,
wherein the one or more proteins forming the shell comprise serum
albumin; wherein the core and the shell each comprise one or more
therapeutic agents; wherein the particle formulation is configured
to independently release the therapeutic agents from the core and
the shell; and wherein the therapeutic agents are configured to be
delivered by active targeting, wherein the active targeting is done
by conjugating the core-shell formulation with transferrin ligand
or EGFR.
2. The formulation of claim 1, wherein the one or more therapeutic
agents of the core comprise doxorubicin and the one or more
therapeutic agents of the shell comprise sorafenib.
3. The formulation of claim 1, wherein the one or more therapeutic
agents of the core comprise sorafenib and the one or more
therapeutic agents of the shell comprise doxorubicin.
4. The formulation of claim 1, wherein the one or more therapeutic
agents of the core comprise dasatinib and the one or more
therapeutic agents of the shell comprise sorafenib.
5. The formulation of claim 1, wherein the one or more therapeutic
agents of the core comprise sorafenib and the one or more
therapeutic agents of the shell comprise dasatinib.
6. The formulation of claim 1, wherein the core is of average size
.ltoreq.500 nm.
7. The formulation of claim 1, wherein the shell is of average
thickness .ltoreq.200 nm.
8. The formulation of claim 1, wherein the therapeutic agents are
configured to be delivered from the shell and core
sequentially.
9. The formulation of claim 1, wherein the therapeutic agents are
configured to be delivered from the shell and core
simultaneously.
10. A core-shell particle formulation for delivering multiple
therapeutic agents comprising: one or more polymers forming a core,
wherein the one or more polymers forming the core comprise poly
vinyl alcohol; and one or more proteins forming a shell
encapsulating the core to form a particle formulation, wherein the
one or more proteins forming the shell comprise protamine; wherein
the core and the shell each comprise one or more therapeutic
agents; wherein the particle formulation is configured to
independently release the therapeutic agents from the core and the
shell; and wherein the therapeutic agents are configured to be
delivered by active targeting, wherein the active targeting is done
by conjugating the core-shell formulation with transferrin ligand
or EGFR.
11. The formulation of claim 10, wherein the one or more
therapeutic agents of the core comprise doxorubicin and the one or
more therapeutic agents of the shell comprise sorafenib.
12. The formulation of claim 10, wherein the one or more
therapeutic agents of the core comprise sorafenib and the one or
more therapeutic agents of the shell comprise doxorubicin.
13. The formulation of claim 10, wherein the one or more
therapeutic agents of the core comprise dasatinib and the one or
more therapeutic agents of the shell comprise sorafenib.
14. The formulation of claim 10, wherein the one or more
therapeutic agents of the core comprise sorafenib and the one or
more therapeutic agents of the shell comprise dasatinib.
15. The formulation of claim 10, wherein the core is of average
size .ltoreq.500 nm.
16. The formulation of claim 10, wherein the shell is of average
thickness .ltoreq.200 nm.
17. The formulation of claim 10, wherein the therapeutic agents are
configured to be delivered from the shell and core
sequentially.
18. The formulation of claim 10, wherein the therapeutic agents are
configured to be delivered from the shell and core simultaneously.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/465,521 filed on Aug. 21, 2014 titled "CORE-SHELL PARTICLE
FORMULATION FOR DELIVERING MULTIPLE THERAPEUTIC AGENTS", 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 a core-shell particle
formulation for delivering multiple therapeutic agents. More
particularly, core-shell particle formulation configured to
independently release therapeutic agents from the core and the
shell. Moreover, the core-shell particle bearing therapeutic agents
enables treatment against the diseases such as cancer, inflammatory
and auto-immune diseases.
BACKGROUND
[0003] Effective treatments for patients with cancer represented a
major challenge in the medical field. The current regimen of
surgical resection, external beam radiation therapy, and systemic
chemotherapy has been partially successful in some kinds of
malignancies. In some malignancies, such as brain malignancies,
this regimen produces a median survival of less than one year.
Though effective in some kinds of cancers, the use of systemic
chemotherapy reached only minor success in the treatment of cancers
of the colon-rectum, esophagus, liver, pancreas, and kidney, and
skin. A major problem with systemic chemotherapy for the treatment
of these types of cancers is that the systemic drug release
required for control over tumor growth cell.
[0004] Efforts to improve delivery of chemotherapeutic agents to
the tumor site have resulted in advances in organ-directed
chemotherapy, for example, by continuous systemic infusion.
However, continuous infusions of anticancer drugs generally have
not shown a clear benefit over pulse or short-term infusions. Some
of the prior arts are as follows,
[0005] US20070053845 discloses a drug delivery system of two
different therapeutic agents by means of a core nanoparticle with
one therapeutic agent and an outer layer coating of the said core
as a shell nanoparticle with second therapeutic agent. The coating
of the therapeutic agent as the outer shell delivers the drugs in
the faster or even in uncontrollable rate, when compared to the
drug delivery from the core.
[0006] WO2007069272 discloses a composition for cancer therapy
comprises nanoparticles of at least one anticancer drug and at
least one polymer. WO2007119601 discloses a pharmaceutical
composition with the nanoparticles of platelet-derived growth
factor (PDGF) receptor tyrosine kinase inhibitor.
[0007] Most of the FDA approved nanoformulations and other drug
delivery systems reported till date are single agent delivery
vehicles which pose structural constraints to encapsulate and
release 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.
[0008] However, 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
therapeutic agents toward targeted diseased sites.
SUMMARY OF THE INVENTION
[0009] A core-shell particle formulation for delivering multiple
therapeutic agents is disclosed. In one aspect the formulation
comprises one or more polymers forming a core and one or more
proteins forming a shell encapsulating the core to form a particle
formulation. In various aspects, the core and the shell each
comprise one or more therapeutic agents and the particle
formulation is configured to independently release therapeutic
agents from the core and the shell. In one aspect, the therapeutic
agents are configured to be delivered by either passive or active
targeting.
[0010] In one aspect, the core is of average size .ltoreq.500 nm
and the shell is of average thickness .ltoreq.200 nm respectively.
In various aspects, the core and shell are loaded with one or more
small molecule kinase inhibitors and chemotherapeutic drugs. In one
aspect, the shell comprises one or both of hydrophilic and
hydrophobic therapeutic agents. The therapeutic agents are
configured to be delivered from the shell and core either
sequentially or simultaneously.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] 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:
[0012] FIG. 1A illustrates a nanoparticle core-shell formulation
according to one embodiment.
[0013] FIG. 1B is a schematic of the method of preparing a
core-shell formulation of the invention.
[0014] FIG. 2 shows an example reaction scheme including steps
involved in the synthesis of the core-shell particle
formulation.
[0015] FIG. 3A shows the scanning electron microscopic (SEM) image
of the PLGA-Everolimus nano-core.
[0016] FIG. 3B shows the SEM image of the final core-shell
nanoconstruct.
[0017] FIG. 3C shows the SEM image of the PLGA-everolimus
core-albumin-sorfenib shell particle formulation.
[0018] FIGS. 4A-4F show the cytotoxicity of free drugs and
nanoformulations of everolimus, sorafenib and combination
core-shell particle formulation in KG1a and PBMC. FIG. 4A shows
cell viability of free everolimus and nano everolimus (1-25 nM)
treated KG1a in comparison.
[0019] FIG. 4B shows cell viability of free everolimus and nano
everolimus (1-25 nM) treated PBMC in comparison.
[0020] FIG. 4C shows cell viability of free sorafenib and nano
sorafenib (0.1-5 .mu.M) treated KG1a in comparison.
[0021] FIG. 4D shows cell viability of free sorafenib and nano
sorafenib (0.1-5 .mu.M) treated PBMC in comparison.
[0022] FIG. 4E shows cell viability of 10 nM everolimus and 1 .mu.M
sorafenib free drug and nanoformulation in KG1a in comparison.
[0023] FIG. 4F shows cell viability of 10 nM everolimus and 1 .mu.M
sorafenib free drug and nanoformulation in PBMC in comparison.
[0024] FIG. 5A shows confocal DIC images of untreated KG1a
cells.
[0025] FIG. 5B shows confocal DIC images of 10 nM everolimus and 1
.mu.M sorafenib nanomedicine treated KG1a cells.
[0026] FIG. 5C shows confocal DIC images of Western blot analysis
of KG1a cells treated with free drug combination and nanomedicine
for 72 hours.
[0027] FIGS. 6A-6F show the mode of cell death induced by the
particle formulation analyzed using flow cytometry and confocal
microscopy. FIG. 6A shows the flow cytometric apoptosis data of the
untreated KG1a cells.
[0028] FIG. 6B shows KG1a cells treated with 10 nM nano
everolimus.
[0029] FIG. 6C shows 1 .mu.M nano sorafenib.
[0030] FIG. 6D shows 10 nM everolimus +1 .mu.M sorafenib particle
formulation using annexin V FITC and PI staining.
[0031] FIGS. 6E and 6F represent the confocal microscopic images
showing both apoptotic and late apoptotic cell fractions.
DETAILED DESCRIPTION
[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 protein, polymer or their combinations, 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] "Polymer-core/polymer-shell and polymer-core/protein-shell
nanomedicine" may refer to nanomedicine constructs comprising a
nano-core formed by one type of polymer loaded with one type of
chemotherapeutic drug and an outer nano-shell formed by another
type of polymer loaded with another drug. Alternatively, the shell
can be formed by a protein.
[0036] Nanomedicine may be formed by encapsulating at least one
therapeutic agent within a biocompatible and biodegradable
polymeric nano-core and encapsulating at least one therapeutic
agent within a biocompatible polymer/protein nano-shell and
connecting the disease targeting ligands to the surface of the
nano-shell. Nanomedicine may have a size of 1-1000 nm. In one
embodiment the nanomedicine has a size of 1-300 nm. The
nanomedicine may be produced in the form of lyophilized powders or
liquid dispersions.
[0037] Therapeutics may be small molecule kinase inhibitors,
chemotherapeutic drugs, prodrugs, etc. that have a therapeutic
effect against diseases including cancer, inflammatory and
auto-immune diseases and the like.
[0038] Small-molecule kinase inhibitors may be synthetic or natural
compounds, typically of a molecular size of less than 1,000 Daltons
that selectively inhibit particular kinases, typically through
ATP-competitive interactions with the catalytic pocket or through
allosteric interactions with other regions of the kinase.
[0039] The term "combinatorial therapy" as used herein refers to
simultaneous use of two or more therapeutics to treat a single
disease.
[0040] The term "targeting ligand" as used herein refers to active
biomolecules that can specifically identify and target an antigen
or receptor on the surface of cell-membrane of cancer cells.
Targeting ligands may include antibodies, peptides, aptamers,
vitamins like folic acid, sugar molecules like mannose,
carbohydrates etc.
[0041] The term "pharmacokinetics" as used herein refers to the
fate of substances administered externally to the body, including
their rate and extent of liberation, absorption, distribution,
metabolism and excretion.
[0042] 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.
[0043] In one embodiment, core-shell particle formulations for
delivering multiple therapeutic agents and methods for their
preparation are disclosed, as shown in FIGS. 1A and 1B,
respectively. As shown in FIG. 1A, in one embodiment, the
formulations of the invention comprise one or more polymers 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. 1B, the formulation is obtained using the steps
shown in the figure. In step 201, nanoparticles comprising one or
more polymers are prepared. In step 202, the core nanoparticles 101
are coated with a therapeutic agent 102 to obtain core nanoparticle
103 loaded with the agent. In step 203, a therapeutic agent 104 is
blended with protein 105 for forming the protein shell. The
drug-loaded core nanoparticles 103 are added to the blended protein
105 in step 204. In step 204, the therapeutic agent 104 is
incorporated into the protein 105 and forms a shell around the core
103. Finally, in step 205, the fully formed core-shell
nanoparticles 110 are separated from solution for therapeutic
use.
[0044] The particle formulation is configured to independently
release therapeutic agents 104 from the core 101 and the shell 102.
The shell 102 encapsulates the core 101 to form a particle
formulation 103. The particle formulations are used for
combinational therapy against the diseases such as cancer,
inflammatory and auto-immune diseases.
[0045] In various embodiments, the polymers for the core 101 are
natural or synthetic biocompatible polymer at least one from the
group, but not limited to poly glycolic acid (PGA),
poly(lactic-co-glycolic acid) (PLGA), glycolide/trimethylene
carbonate copolymers (PGA/TMC), poly-lactides (PLA), poly-L lactide
(PLLA), poly-DL-lactide (PDLLA), L-lactide/DL-lactide copolymers,
lactide/tetramethyl-glycolide copolymers, poly-caprolactone (PCL),
poly-valerolacton (PVL), poly-hydroxy butyrate (PHB), poly vinyl
alcohol (PVA) poly-hydroxyvalerate (PHV), polyvinylpyrrolidone
(PVP), polyethyleneimine (PEI) and lactide/trimethylene carbonate
copolymers, chitosan, carboxymethyl chitosan, chitin, pollulan,
etc., or blends thereof.
[0046] In various embodiments, the protein 105 forming the 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.
[0047] In various embodiments the polymer core 101 is formed by a
method that is one of spontaneous emulsification, solvent
diffusion, salting out, emulsification-diffusion, micro emulsion,
double microemulsion, ultrasonication, nano-precipitation or
electrospray. In various embodiments of the method shown in FIG.
1B, the protein shell 106 is formed over the polymer core by simple
desolvation, co-acervation, complex co-acervation,
nano-precipitation, sol-gel processing, spray drying, salting-out
or cross linking and the like. In some embodiments, the prepared
core-shell nanomedicine 110 is purified by centrifugation and
lyophilisation.
[0048] In one embodiment, the core 101 is of average size
.ltoreq.500 nm and the shell 106 is of average thickness
.ltoreq.200 nm respectively. In various embodiments, the core and
shell are loaded with one or more small molecule kinase inhibitors
and chemotherapeutic drugs. In one embodiment, the shell comprises
either hydrophilic or hydrophobic therapeutic agents, or both types
of agents.
[0049] In one embodiment, the small molecule kinase inhibitor is
chosen from: the inhibitors of tyrosine kinase including epidermal
growth factor receptor inhibitors such as erlotinib, lapatinib,
neratinib, gefitinib, mubritinib, afatinib, pelitinib, vandetenib,
vascular endothelial growth factor receptor and platelet derived
growth factor receptor inhibitors such as brivanib, axitinib,
tivozanib, cedivanib, crenalonib, dovitinib, foretinib, linifanib,
masitinib, motesanib, pazopanib, ponatinib, regorafenib, fibroblast
growth factor receptor inhibitors such as danusertib, PD173074,
vargatef, Rous sarcoma oncogene/breakpoint cluster region/Abl
inhibitors such as dasatinib, bafetinib, nilotinib, sophoretin,
saracatinib, PP121, fingolimod, AT9283, insulin-like growth factor
1 receptor inhibitors such as BMS-536924, BMS-554417, BMS-754807,
GSK-1838705A, NVP-ADW742, NVP-AEW541, OSI-906, FLT-3 inhibitors
such as cabozantinib, quizartinib, KW 2449, HER-2 inhibitors such
as caneratinib, AEE788, BIBW22992, CP-724714, c-Kit such as
imatinib, Ki8751, MP-470, OSI-930, telatinib, c-Met such as
SUII274, SGX-532, PHA-665752, PF-2341066, PF-04217903, MGCD-265,
JNJ-38877605, AMG-208, ALK inhibitors such as LDN-193189,
SB-525334, TAE-684, ETA receptor inhibitors such as zibotentan, HIF
inhibitors such as 2-methoxyestradiol, Syk inhibitors such as R406,
R788, fostamatinib, Tie2 kinase inhibitors such as XL-184, Vascular
disrupting agents such as plinabulin, DMXAA, cell cycle/check point
inhibitors like polo-like kinase (PLK) inhibitors such as
volasertib, BI-2536, BI60727, GSK-461364, HMN-214, ON-01910, cyclin
dependent kinase (CDK) inhibitors such as seliciclib, indirubin,
flavopiridol, BS-181, AT-7519, PHA-793887, R547, topoisomerase
inhibitors such as adriamycin, camptothecin, etoposide, idarubicin,
irinotecan, topotecan, mitoxantrone, microtubule inhibitors such as
docetaxel, paclitaxel, vincristine, antimetabolites such as
decitabine, gemcitabine, fludarabine, telomerase inhibitors such as
BIBR 1532, DNA & RNA replication inhibitors such as
clarithromycin, cytarabine, mitoxantrone HCl, dihydrofolate
reductase inhibitors such as NSC-131463, methotrexate, HDAC
inhibitors such as droxinostat, givinostat, belinostat, vorinostat,
panobinostat, mocetinostat, entinostat, valproic acid, Bcl-2
inhibitors such as navitoclax, obatoclax, ABT 737 and TNF-a
inhibitors such as lenalidomide, pomalidomide, p53 inhibitors such
as JNJ 26854165, NSC 207895, PARP inhibitors such as BSI-201,
INO-1001, MK-4827, veliparib, olaparib, MAPK inhibitors such as
AS-703026, PD98059, PD0325901, JTP-74057, U0126, GDC-0879, ZM
336372, SP600125, selumatinib, vemurafenib, sorafenib, tipifarnib,
PI3K/Akt/mTOR inhibitors such as acadesine, A66, CAL101, BEZ235,
GDC-0941, Phenformin, PI-103, quercetin, PP121, XL765, XL147,
everolimus, deforolimus, chrysophanic acid, temsirolimus,
rapamycin, perifosine, triciribine, integrase and protease
inhibitors such as elvitegravir, raltegravir, atazanavir,
bortezomib, ritonavir, Wnt/Hedgehog/Notch inhibitors such as
cyclopamine, vismodegib, semagacestat, BMS-708163, ICG-001,
XAV-939, Jak/STAT inhibitors such as tofacitinib, ruxolitinib,
cryptotanshinone, NSC-74859, AZ-960, AG-490, PKC inhibitors such as
zoledronic acid, enzastaurin, chelerythrine, TGF-P inhibitors such
as LY2157299, SB431542, antioxidant inhibitors such as
diethyl-dithiocarbamate, methoxyestradiol, 1-buthionine
sulfoximine, 3-amino-1,2,4-triazole or combinations thereof.
[0050] In various embodiments, the chemotherapeutic drug is chosen
from the group of anti-neoplastic agents such as aminoglutethimide,
amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin,
buserelin, busulfan, campothecin, capecitabine, carboplatin,
carmustine, chlorambucil, cisplatin, cladribine, clodronate,
colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine,
dactinomycin, daunorubicin, dienestrol, diethylstilbestrol,
docetaxel, doxorubicin, epirubicin, estradiol, estramustine,
etoposide, exemestane, filgrastim, fludarabine, fludrocortisone,
fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein,
goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib,
interferon, irinotecan, ironotecan, letrozole, leucovorin,
leuprolide, levamisole, lomustine, mechlorethamine,
medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
nocodazole, octreotide, oxaliplatin, paclitaxel, pam idronate,
pentostatin, plicamycin, porfimer, procarbazine, raltitrexed,
rituximab, streptozocin, suramin, tamoxifen, temozolomide,
teniposide, testosterone, thioguanine, thiotepa, titanocene
dichloride, topotecan, trastuzumab, tretinoin, vinblastine,
vincristine, vindesine, vinorelbine and combinations thereof. In
various embodiments, the therapeutic agents 102 and 104 are
configured to be delivered 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,
transferrin receptors and like, peptides such as R.GD, CRGD, LyP-1,
bombesin (BBN), FSH33, truncated human basic fibroblast growth
factor (tbFGF), octreotide, small molecules such as folic acid,
mannose, hyaluronic acid (HA), proteins such as transferrin,
somatostatin or aptamers. In one embodiment, the therapeutic agents
are configured to be delivered from the shell and core either
sequentially or simultaneously.
[0051] Thus, the drug delivery system for delivering combination
therapies is achieved as each agent provides the desired maximal
effect, independently and without interference. The multi targeted
nanoparticle formulation results enhanced anti-cancer activity
compared to single drug loaded nanoparticles. Moreover, core-shell
construct can be targeted to the diseased site by conjugating with
a wide array of cancer targeting ligands and monoclonal antibodies
against cancer cell-specific surface antigens includes folic acid,
transferrin, and monoclonal antibodies against CD123, CD33, CD47,
CLL-1, etc. Such a system would be useful not only in the treatment
of cancer but would also find use in the treatment of other
diseases such as autoimmune disease (e.g., rheumatoid arthritis),
inflammatory diseases (e.g., asthma), neurological diseases (e.g.,
epilepsy), and ophthalmological diseases (e.g., diabetic
retinopathy). Therefore, a core-shell platform developed for
sequential and simultaneous delivery of the loaded drugs depending
on the nature of the construct such as polymer and protein, its
molecular weight, degradation kinetics and nature of drug
binding.
[0052] The invention is further explained in 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
[0053] In this example, preparation of a combinatorial
polymer-protein core-shell particle formulation as shown
schematically in FIG. 2 with a mTOR small molecule kinase
inhibitor, everolimus loaded polymeric [PLGA: poly
(lactic-co-glycolic acid (50:50)] nanocore and a small molecule
multi-kinase inhibitor sorafenib entrapped protein shell is
presented. 1 mg of everolimus was dissolved in 5 ml 2.5% w/v
PLGA-acetone solution and allowed to incubate overnight with
continuous stirring at a speed of 500 rpm at 4.degree. C.
Emulsification of the polymer-drug solution was achieved by the
drop-wise addition of the above solution into 5 ml distilled water
containing 0.4% v/v pluronic F-127, with continuous stirring at a
speed of 1500 rpm on a magnetic stirrer. Acetone was evaporated out
from the o/w emulsion yielding a colloidal dispersion of everolimus
loaded PLGA nanoparticles.
[0054] The nanoparticles were then recovered from the solution by
centrifugation at 5000 rpm for 10 minutes. The harvested
nanoparticles were washed with distilled water and the final pellet
was resuspended in 5 ml distilled water and lyophilized for 48 h to
yield freeze-dried PLGA-everolimus. Albumin-sorafenib was prepared
by nano-precipitation wherein briefly, 5 mg of BSA was dissolved in
5 ml of double-distilled water. To this, 64 .mu.L of 15.7 mM
DMSO-sorafenib was added drop wise with continuous stirring at a
speed of 1500 rpm on a magnetic stirrer. To the resulting colloidal
solution, 10 mg of EDC was added and incubated in the dark at
4.degree. C. with continuous stirring (500 rpm). The
nano-dispersion of albumin-sorafenib was stored at 4.degree. C. and
used as synthesized for further characterization and cell culture
studies.
Example-2
[0055] In this example, preparation of a combinatorial
polymer-polymer core-shell particle formulation with a
chemotherapeutic drug, paclitaxel loaded polycaprolactone (PCL)
nano-core and dasatinib encapsulated chitosan shell is presented. 1
mg Paclitaxel was dissolved in 5 ml 1.0 wt PCL solution in
chloroform and allowed to incubate overnight with continuous
stirring at a speed of 500 rpm at 4.degree. C. Emulsification of
the polymer-drug solution was achieved by the drop-wise addition of
the above solution into 5 ml distilled water containing 0.4% v/v
pluronic F-127, with continuous stirring at a speed of 1500 rpm on
a magnetic stirrer. Chloroform was evaporated out from the o/w
emulsion yielding a colloidal dispersion of paclitaxel loaded PCL
nanoparticles. The nanoparticles were then recovered from the
solution by centrifugation at 5000 rpm for 10 minutes. The
harvested nanoparticles were washed with distilled water and the
final pellet was resuspended in 5 ml distilled water and
lyophilized for 48 h to yield freeze-dried PCL-paclitaxel
nanoparticles.
[0056] The lyophilized particles are then mixed with 0.5% chitosan
solution containing 5 mM sorafenib. 0.25 wt % Tween 80 was added to
this solution to prevent particle aggregation and the system was
subjected to stirring for 30 min. The chitosan-sorafenib nanoshell
was prepared over PCL-paclitaxel nanoparticles by ionic gelation
process. Aqueous tripolyphosphate (TPP: 0.25% w/v) solution was
added drop wise into the above solution and stirred under room
temperature. The core shell nanoparticles were obtained by
centrifuging the suspension at 12,000 rpm for 30 min.
Example-3
[0057] The size and morphology of the core-shell particle
formulation was characterized using dynamic light scattering
technique and electron microscopy. In FIG. 3A, the nano-everolimus
polymeric core nanoparticle exhibited smooth and regular spherical
shape with average size of .about.280 nm as observed in scanning
electron microscopy (SEM) analysis. The dynamic light scattering
measurements also exhibited mean diameter of .about.284.+-.20 nm.
Zeta potential analysis, revealed an average potential of -15.42
mV, indicative of a good stable dispersion of nano-everolimus in an
aqueous medium. Everolimus was efficiently loaded into PLGA
nanoparticles attaining an encapsulation efficiency of
94.58.+-.2.56%.
[0058] The loading efficiency of sorafenib in albumin shell was
.about.95%, owing to strong hydrophobic interactions. In FIG. 3B,
the SEM images of the final core-shell nanoconstruct indicated an
increase in the particle size to .about.330 nm, compared to that of
nano-core. DLS analysis indicated average particle size of 335
nm.+-.12.6 nm which is in line with SEM analysis which recorded
size of the construct to be .about.345 nm. The field emission
transmission electron microscopic image of a single nano-construct
clearly revealed the formation of core-shell structure where
electron-dense PLGA-everolimus nano-core was found decorated with a
thin (25-30 nm) shell of albumin-sorafenib. Furthermore, the
particle formulation suspension exhibited a zeta potential of
-10.86 mV. FIG. 3C shows the SEM image of the PLGA-everolimus
core-albumin-sorfenib shell particle formulation.
Example-4
[0059] In FIG. 4, the cytotoxic potential of the core-shell
particle formulation was investigated in Acute Myeloid Leukemia
primitive cell line, KG1a, and human peripheral blood derived
mononuclear cells, PBMC. To optimize the concentration of the small
molecules in core-shell particle formulation, the dose-response of
everolimus and sorafenib in KG1a and PBMC was identified. To
determine the cytotoxicity of free drugs and nanoformulations, both
KG1a and PBMC were treated with respective concentration ranges of
everolimus and sorafenib for 72 hours. Both free drugs and
nanoformulations of everolimus and sorafenib were shown to exhibit
similar toxicity profiles. Free everolimus and nano-everolimus did
not exert any significant cytotoxicity towards KG1a, over a
concentration range from 1 to 25 nM, which demonstrated its
inefficiency as a monotherapy agent in FIG. 4A. However, both free
and nano-everolimus showed slight toxicity towards PBMC in FIG. 4B
which nevertheless falls within the tolerable limit. This toxicity
of everolimus might account from its immunosuppressive properties.
In contrast, sorafenib demonstrated concentration dependent
toxicity in KG1a, over a concentration range of 0.1 to 5 .mu.M,
while causing only minimal toxicity towards PBMC in FIG. 3C, which
could be projected as an ideal anti-cancer agent. However, it is
clear from FIG. 3D that sorafenib too cannot manifest as an
efficient single agent against AML, since .about.40% of cells
remain viable even after treating them with 5 .mu.M sorafenib for
72 h. Therefore, possible synergistic toxicity by treating cells
with a combination of both everolimus and sorafenib are
investigated. For this, initially identified the dose-response of
currently used cytotoxic drug combination, Ara-C and daunorubicin,
replicating the clinically administered concentration ratios under
in vitro conditions in KG1a and PBMC. The toxicity level of the 100
nM Ara-C+50 nM daunorubicin was taken as reference for anti-cancer
efficacy of nanoformulation, as it ideally represents the
clinically administered ratio of both chemotherapeutic drugs.
Sub-IC50 concentrations of both everolimus and sorafenib were
tested in combination for possible synergism, aiming to further
lower the concentration of individual drugs. Therefore in the
subsequent set of experiments, KG1a cells were treated with free
drug combinations of 10 nM everolimus and 1 .mu.M sorafenib and the
core-shell particle formulation encapsulating same concentration of
drugs along with the chemodrug combination.
[0060] FIG. 4E shows the toxicity exerted by the synergizing
combination of drugs. Surprisingly, the cytotoxic profile of the
kinase inhibitor combination proved to be as effective as the
chemotherapeutic combination. The 10 nM everolimus-1 .mu.M
sorafenib free drug combination exerted a toxicity of .about.71%
compared to A100+D50 chemotherapeutic combination toxicity of
.about.72%. Whereas, the core-shell particle formulation
encapsulating 10 nM everolimus-1 .mu.M sorafenib demonstrated
maximal toxicity of the lot, registering .about.75% toxicity.
Further, from FIG. 4F shows that the 10 nM everolimus-1 .mu.M
sorafenib free drug combination and particle formulation exerted
minimal toxicity of .about.20% towards PBMC, in comparison to
lethal toxicity of .about.80%, exerted by the chemotherapeutic
combination.
[0061] The most striking observation from the above results is
regarding the excellent synergy exhibited by the combination of
sub-IC50 concentrations of everolimus and sorafenib. .about.70% of
cells treated with 10 nM everolimus and .about.68% cells treated
with sorafenib remained viable after 72 hours of incubation.
Whereas, only 25% cells survived the treatment with the kinase
inhibitor combination. In FIGS. 4G and 4H, subsequent experiments
using combinations of 25 nM everolimus and 5 .mu.M sorafenib proved
to be extremely cytotoxic towards KG1a leaving a 5% surviving
population whereas .about.74% PBMCs survived the kinase
combinatorial treatment, as opposed to .about.5% PBMC that survived
the corresponding 250 nM Ara-C+125 nM daunorubicin chemotherapeutic
treatment.
Example-5
[0062] The morphological characterization and western blot analysis
of primitive AML cell lines treated with the core-shell particle
formulation were analyzed. The particle formulation exerted evident
morphological changes and loss of membrane integrity compared to
same concentrations of free everolimus and free sorafenib as seen
from the confocal DIC image in FIGS. 5A and B. To analyze the
extent of mTOR inhibition by everolimus and induction of apoptosis
by sorafenib, the phosphorylation status of p70S6 kinase, and Mcl-1
was determined in KG1a treated with the free drug combination and
core-shell particle formulation. Both free drug combinations and
free drugs were found to attenuate mTOR signaling and reduced the
expression level of phospho-p70S6K in FIG. 5C. The observed
reduction in phospho-p70S6K was more pronounced in the nano
everolimus treated KG1a cells indicating an increasingly efficient
delivery of nano everolimus to the cells over the free drug
formulation. Similarly, the downregulation of both Mcl-1 and pSTAT5
in both free drug and particle formulation treated KG1a was
confirmed through Western blot analysis.
Example-6
[0063] In another embodiment of the said method, mode of cell death
induced by the particle formulation was analyzed using flow
cytometry and confocal microscopy in FIG. 6. The cells treated with
nano-everolimus, nano-sorafenib and core-shell medicine were
stained with FITC conjugated annexin-V and PI and the corresponding
flow data and confocal microscopic images shows both apoptotic and
late-apoptotic cell fractions in FIGS. 6A, 6B, 6C and 6D.
[0064] Primarily, the invention represents a polymer-protein
core-shell particle formulation nanoparticle that aids
sequential/simultaneous delivery of at least two small molecule
kinase inhibitors. Specifically, the core-shell construct is based
on a polymeric core made of poly-lactide-co-glycolide co-polymer
nanoparticle encapsulating an mTOR inhibitor, everolimus and an
albumin nano-shell encapsulating a multi-kinase inhibitor,
sorafenib. Moreover, the invention comprises the highly hydrophobic
payloads which are incorporated within the polymeric and protein
matrix significantly improves therapeutic outcome by enhancing the
aqueous solubility, dissolution rate and enhanced uptake by cancer
cell specific targeting. The targeting efficacy of the
nanoformulation can be achieved by conjugating with a wide array of
cancer targeting ligands and monoclonal antibodies against cancer
cell-specific surface antigens; the examples of which include folic
acid, transferrin, and monoclonal antibodies against CD123, CD33,
CD47, CLL-1, etc.
* * * * *