U.S. patent application number 12/485818 was filed with the patent office on 2010-04-29 for methods for the preparation of targeting agent functionalized diblock copolymers for use in fabrication of therapeutic targeted nanoparticles.
This patent application is currently assigned to BIND BIOSCIENCES, INC.. Invention is credited to Mir M. Ali, Jeff Hrkach, Stephen E. Zale.
Application Number | 20100104645 12/485818 |
Document ID | / |
Family ID | 41507667 |
Filed Date | 2010-04-29 |
United States Patent
Application |
20100104645 |
Kind Code |
A1 |
Ali; Mir M. ; et
al. |
April 29, 2010 |
METHODS FOR THE PREPARATION OF TARGETING AGENT FUNCTIONALIZED
DIBLOCK COPOLYMERS FOR USE IN FABRICATION OF THERAPEUTIC TARGETED
NANOPARTICLES
Abstract
This application provides methods of making nanoparticles using
pre-functionalized poly(ethylene glycol) (also referred to as PEG)
as a macroinitiator for the synthesis of diblock copolymers. These
diblock copolymers comprise a poly(ethylene glycol) block bearing a
targeting agent at its terminus and a second biocompatible and
biodegradable hydrophobic polymer block (e.g. a poly(ester)). The
poly(ethylene glycol) is hetero-bifunctional with a targeting
moiety (agent) covalently bound to its .alpha. terminus and a
polymerization initiating functional group (e.g., a hydroxyl group)
present on its .omega. terminus. Ring opening polymerization yields
the desired poly(ester)-poly(ethylene glycol)-targeting agent
polymer that is used to impart targeting capability to therapeutic
nanoparticles. This "polymerization from" approach typically
employs precursors of the targeting agent wherein the reactivity of
functional groups of the targeting agent is masked using protecting
groups. Also described is a "coupling to" that utilized the
poly(ethylene glycol)-targeting agent conjugate where the targeting
agent remains in its native un-protected form. This method uses
"orthogonal" chemistry that exhibit no cross reactivity towards
functional groups typically found within targeting agents of
interest. Nanoparticles produced according to the disclosed methods
as well as their use in the treatment of various diseases are also
provided.
Inventors: |
Ali; Mir M.; (Woburn,
MA) ; Hrkach; Jeff; (Lexington, MA) ; Zale;
Stephen E.; (Hopkinton, MA) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO Box 142950
GAINESVILLE
FL
32614
US
|
Assignee: |
BIND BIOSCIENCES, INC.
Cambridge
MA
|
Family ID: |
41507667 |
Appl. No.: |
12/485818 |
Filed: |
June 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61061712 |
Jun 16, 2008 |
|
|
|
Current U.S.
Class: |
424/489 |
Current CPC
Class: |
A61K 47/6911 20170801;
A61P 29/00 20180101; A61K 47/34 20130101; A61P 31/12 20180101; A61P
31/04 20180101; A61K 47/60 20170801; A61K 9/5153 20130101; A61P
35/00 20180101 |
Class at
Publication: |
424/489 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of preparing a nanoparticle comprising: providing a
targeting agent; providing a functionalized poly(ethylene glycol)
(PEG) polymer; providing a targeting agent ligand; reacting the
functionalized poly(ethylene glycol) polymer with the targeting
agent to form a targeting agent-PEG polymer complex; and mixing the
targeting agent-PEG polymer complex with a second polymer and a
therapeutic agent to form a nanoparticle.
2. The method of claim 1, wherein the poly(ethylene glycol) is
hetero-bifunctional and said targeting agent is covalently bound to
the .alpha. terminus of said poly(ethylene glycol) and at least one
polymerization initiating functional group is present on the
.omega. terminus of said poly(ethylene glycol).
3. The method of claim 2, wherein said at least one polymerization
initiating functional group is a hydroxyl (--OH) group or an amine
(--NH.sub.2) group at the free .omega. terminus that reacts with a
second copolymer.
4. The method of claim 3, wherein the second polymer comprises a
blend of two or more polymers and contains at least one functional
group that reacts the functional group present at the free .omega.
terminus of said poly(ethylene glycol) and said at least one
functional group of said blend of two or more polymers is a
hydroxyl group, a NHS group or an amine group.
5. The method of claim 1, wherein the second polymer or the
copolymer is a polyester copolymer that contains at least one
functional group selected from a hydroxyl group, a NHS group or an
amine group and that reacts the functional group present at the
free .omega. terminus of said poly(ethylene glycol).
6. The method of claim 4, wherein the second polymer or the
copolymer is a polyester copolymer that contains at least one
functional group selected from a hydroxyl group, a NHS group or an
amine group and that reacts the functional group present at the
free .omega. terminus of said poly(ethylene glycol).
7. The method of claim 6, wherein said polyester copolymer
comprises a heteropolymer or a homopolymer.
8. The method of claim 7, wherein said heteropolymer comprises
lactic acid and glycolic acid units or poly(lactic acid-co-glycolic
acid) and poly(lactide-co-glycolide) units (PLGA); and said
homopolymer comprises glycolic acid units (PGA), lactic acid units
(PLA), poly-L-lactic acid units, poly-D-lactic acid units,
poly-D,L-lactic acid units, poly-L-lactide units, poly-D-lactide
units or poly-D,L-lactide units.
9. The method of claim 6, wherein said polyester copolymer is
selected from polyhydroxyacids; PEGylated polymers and copolymers
of lactide units and glycolide units, PEGylated PLA, PEGylated PGA,
PEGylated PLGA, polyanhydrides, poly(ortho ester) PEGylated
poly(ortho ester), poly(caprolactone), PEGylated
poly(caprolactone), polylysine, PEGylated polylysine, poly(ethylene
inline), PEGylated poly(ethylene imine),
poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester), poly[a-(4-aminobutyl)-L-glycolic
acid] or derivatives thereof.
10. The method of claim 1, wherein free carboxylic acid groups or
free hydroxyl groups on said targeting agent are protected prior to
reacting the functionalized poly(ethylene glycol) polymer with the
targeting agent to form a targeting agent-PEG polymer complex.
11. The method of claim 4, wherein said functional group of said
second polymer is an amine group that reacts with a hydroxyl group
or carboxylic acid group on said targeting agent-PEG complex.
12. The method of claim 4, wherein said functional group of said
second polymer is a hydroxyl group or an NHS group that reacts with
an amine group on said targeting agent-PEG complex.
13. The method of claim 6, wherein said functional group of said
second polymer or said copolymer is an amine group that reacts with
a hydroxyl group or carboxylic acid group on said targeting
agent-PEG complex.
14. The method of claim 6, wherein said functional group of said
second polymer or said copolymer is a NHS or hydroxyl group that
reacts with an amine group on said targeting agent-PEG complex.
15. The method of claim 1, wherein said second polymer is a blend
of at least two polymers which can be the same or different
polymer, wherein the first of said at least two polymers contains
at least one hydroxyl group or an NHS group as a functional group
and the second of said at least two polymers contains at least one
amine group as said functional group.
16. The method of claim 1, wherein said therapeutic agent is an
antibiotic, anti-cancer agent, antiviral agent, anti-inflammatory
agent a diagnostic agent, a vaccine antigen or a nutraceutical.
17. The method of claim 16, wherein said therapeutic agent is/are
penicillins, aminopenicillins, penicillins in conjunction with
penicillinase inhibitor and/or anti-fungal agents, cephalosporins,
cephamycins, carbapenems, fluoroquinolones, tetracyclines,
macrolides, aminoglycosides, erythromycin, bacitracin zinc,
polymyxin, polymyxin B sulfates, neomycin, gentamycin, tobramycin,
gramicidin, ciprofloxacin, trimethoprim, ofloxacin, levofloxacin,
gatifloxacin, moxifloxacin, norfloxacin, sodium sulfacetamide,
chloramphenicol, tetracycline, azithromycin, clarithyromycin,
trimethoprim sulfate, bacitracin, corticosteroids, medrysone,
prednisolone, prednisolone acetate, prednisolone sodium phosphate,
fluormetholone, dexamethasone, dexamethasone sodium phosphate,
betamethasone, fluorometholone, antazoline, fluorometholone
acetate, rimexolone, loteprednol etabonate, diclofenac (diclofenac
sodium), ketorolac, ketorolac tromethamine, hydrocortisone,
bromfenac, flurbiprofen, antazoline, xylometazoline, cromolyn
sodium, lodoxamide tromethamine, olopatadine HCl, nedocromil
sodium, ketotifen fumurate, levocabastine HCL, azelastine HCL,
pemirolast (pemirolast potassium), epinastine HCL, naphazoline HCL,
emedastine, antazoline, pheniramine, sodium cromoglycate,
N-acetyl-aspartyl glutamic acid, amlexanox, 5-fluorouracil (5-FU),
CPT-11, 10-hydroxy-7-ethylcamptothecin (SN38), S-I capecitabine,
ftorafur, 5'deoxyfluorouridine, UFT, eniluracil, deoxycytidine,
5-azacytosine, 5-azadeoxycytosine, allopurinol, 2-chloroadenosine,
aminopterin, methylene-10-deazaminopterin (MDAM), oxaplatin,
picoplatin, tetraplatin, satraplatin, platinum-DACH, ormaplatin,
CI-973, JM-216, and analogs thereof, 9-aminocamptothecin,
10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin,
TAS 103, L-phenylalanine mustard, ifosphamidemefosphamide,
trophosphamide carmustine, epothilones A-E, tomudex,
6-mercaptopurine, 6-thioguanine, karenitecin, acyclovir,
valacyclovir, ganciclovir, amantadine, rimantadine, lamivudine,
zidovudine, bevacizumab, trastuzumab, rituximab,20-epi-1.alpha., 25
dihydroxyvitamin D3, 4-ipomeanol, 5-ethynyluracil, 9-dihydrotaxol,
abiraterone, acivicin, aclarubicin, acodazole hydrochloride,
acronine, acylfiilvene, adecypenol, adozelesin, aldesleukin, all-tk
antagonists, altretamine, ambamustine, ambomycin, ametantrone
acetate, amidox, amifostine, aminoglutethimide, aminolevulinic
acid, amrubicin, amsacrine, anagrelide, anastrozole,
andrographolide, angiogenesis inhibitors, antagonist D, antagonist
G, antarelix, anthramycin, anti-dorsalizing morphogenetic
protein-1, antiestrogen, antineoplaston, antisense
oligonucleotides, aphidicolin glycinate, apoptosis gene modulators,
apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine
deaminase, asparaginase, asperlin, asulacrine, atamestane,
atrimustine, axinastatin 1, axinastatin 2, axinastatin 3,
azacitidine, azasetron, azatoxin, azatyrosine, azetepa, azotomycin,
baccatin III derivatives, balanol, batimastat, benzochlorins,
benzodepa, benzoylstaurosporine, beta lactam derivatives,
beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor,
bicalutamide, bisantrene, bisantrene hydrochloride,
bisazuidinylspermine, bisnafide, bisnafide dimesylate, bistratene
A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists,
breflate, brequinar sodium, bropirimine, budotitane, busulfan,
buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C,
calusterone, camptothecin derivatives, canarypox IL-2,
capecitabine, caraceraide, carbetimer, carboplatin,
carboxamide-amino-triazole, carboxyamidotriazole, carest M3,
carmustine, earn 700, cartilage derived inhibitor, carubicin
hydrochloride, carzelesin, casein kinase inhibitors,
castanosperrnine, cecropin B, cedefingol, cetrorelix, chlorambucil,
chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin,
cisplatin, cis-porphyrin, cladribine, clomifene analogs,
clotrimazole, collismycin A, collismycin B, combretastatin A4,
combretastatin analog, conagenin, crambescidin 816, crisnatol,
crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives,
curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam,
cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin
hydrochloride, decitabine, dehydrodidemnin B, deslorelin,
dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil,
dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox,
diethyhiorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl
spiromustine, docetaxel, docosanol, dolasetron, doxifluridine,
doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene
citrate, dromostanolone propionate, dronabinol, duazomycin,
duocannycin SA, ebselen, ecomustine, edatrexate, edelfosine,
edrecolomab, eflomithine, eflomithine hydrochloride, elemene,
elsarnitrucin, emitefur, enloplatin, enpromate, epipropidine,
epirubicin, epirubicin hydrochloride, epristeride, erbulozole,
erythrocyte gene therapy vector system, esorubicin hydrochloride,
estramustine, estramustine analog, estramustine phosphate sodium,
estrogen agonists, estrogen antagonists, etanidazole, etoposide,
etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole
hydrochloride, fazarabine, fenretinide, filgrastim, finasteride,
flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,
fludarabine phosphate, fluorodaunorunicin hydrochloride,
fluorouracil, fluorocitabine, forfenimex, formestane, fosquidone,
fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin,
gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors,
gemcitabine, gemcitabine hydrochloride, glutathione inhibitors,
hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea,
hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride,
idoxifene, idramantone, ifosfamide, ihnofosine, ilomastat,
imidazoacridones, imiquimod, immunostimulant peptides, insulin-like
growth factor-1 receptor inhibitor, interferon agonists, interferon
alpha-2A, interferon alpha-2B, interferon alpha-N1, interferon
alpha-N3, interferon beta-IA, interferon gamma-IB, interferons,
interleukins, iobenguane, iododoxorubicin, iproplatm, irinotecan,
irinotecan hydrochloride, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-N triacetate, lanreotide, lanreotide acetate,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,
leukemia inhibiting factor, leukocyte alpha interferon, leuprolide
acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole,
liarozole, liarozole hydrochloride, linear polyamine analog,
lipophilic disaccharide peptide, lipophilic platinum compounds,
lissoclinamide, lobaplatin, lombricine, lometrexol, lometrexol
sodium, lomustine, lonidamine, losoxantrone, losoxantrone
hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium
texaphyrin lysofylline, lytic peptides, maitansine, mannostatin A,
marimastat, masoprocol, maspin, matrilysin inhibitors, matrix
metalloproteinase inhibitors, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, merbarone, mercaptopurine, meterelin, methioninase,
methotrexate, methotrexate sodium, metoclopramide, metoprine,
meturedepa, macroalgal protein kinase C uihibitors, MIF inhibitor,
mifepristone, miltefosine, mirimostim, mismatched double stranded
RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone,
mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide,
mitosper, mitotane, mitotoxin fibroblast growth factor-saporin,
mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid a/myobacterium cell wall SK, mopidamol, multiple drug
resistance gene inhibitor, mustard anticancer agent, mycaperoxide
B, mycobacterial cell wall extract, mycophenolic acid, myriaporone,
n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine,
napavin, naphterpin, nartograstim, nedaplatin, nemorubicin,
neridronic acid, neutral endopeptidase, nilutamide, nisamycin,
nitric oxide modulators, nitroxide antioxidant, nitrullyn,
nocodazole, nogalamycin, n-substituted benzamides,
O6-benzylguanine, octreotide, okicenone, oligonucleotides,
onapristone, ondansetron, oracin, oral cytokine inducer,
ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran,
paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine,
palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,
parabactin, pazelliptine, pegaspargase, peldesine, peliomycin,
pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole,
peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin,
piritrexim, piroxantrone hydrochloride, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, plicamycin, plomestane,
porfimer sodium, porfiromycin, prednimustine, procarbazine
hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic
carcinoma antiandrogen, proteasome inhibitors, protein A-based
immune modulator, protein kinase C inhibitor, protein tyrosine
phosphatase inhibitors, purine nucleoside phosphorylase inhibitors,
puromycin, puromycin hydrochloride, purpurins, pyrazorurin,
pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene
conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl
protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor,
retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin,
riboprine, ribozymes, RH retinamide, RNAi, rogletimide, rohitukine,
romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, safingol
hydrochloride, saintopin, sarcnu, sarcophytol A, sargramostim, SDI1
mimetics, semustine, senescence derived inhibitor 1, sense
oligonucleotides, signal transduction inhibitors, signal
transduction modulators, simtrazene, single chain antigen binding
protein, sizofuran, sobuzoxane, sodium borocaptate, sodium
phenylacetate, solverol, somatomedin binding protein, sonermin,
sparfosafe sodium, sparfosic acid, sparsomycin, spicamycin D,
spirogermanium hydrochloride, spiromustine, spiroplatin,
splenopentin, spongistatin 1, squalamine, stem cell inhibitor,
stem-cell division inhibitors, stipiamide, streptonigrin,
streptozocin, stromelysin inhibitors, sulfinosine, sulofenur,
superactive vasoactive intestinal peptide antagonist, suradista,
suramin, swainsonine, synthetic glycosaminoglycans, talisomycin,
tallimustine, tamoxifen methiodide, tauromustine, tazarotene,
tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors,
teloxantrone hydrochloride, temoporfin, temozolomide, teniposide,
teroxirone, testolactone, tetrachlorodecaoxide, tetrazomine,
thaliblastine, thalidomide, thiamiprine, thiocoraline, thioguanine,
thiotepa, thrombopoietin, thrombopoietin mimetic, thymalfasin,
thymopoietin receptor agonist, thymotrinan, thyroid stimulating
hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine,
titanocene dichloride, topotecan hydrochloride, topsentin,
toremifene, toremifene citrate, totipotent stem cell factor,
translation inhibitors, trestolone acetate, tretinoin,
triacetyluridine, triciribine, triciribine phosphate, trimetrexate,
trimetrexate glucuronate, triptorelin, tropisetron, tubulozole
hydrochloride, turosteride, tyrosine kinase inhibitors,
tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, velaresol, veramine,
verdins, verteporfin, vinblastine sulfate, vincristine sulfate,
vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate
sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate,
vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin,
vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin
stimalamer, or zorubicin hydrochloride, siRNA or any combinations
thereof.
18. The method of claim 1, wherein said at least one targeting
agent is selected from antibodies, PMSA ligands and polypeptides
that bind to epidermal growth factor receptor (EGFR), somatostatin
receptor (SSTR), insulin-like growth factor receptor, folic
acid-receptor, HER2 receptor, interleukin-13 receptor,
gastrin-releasing peptide receptor, CD30, vasoactive intestinal
peptide receptor, gastrin receptor, prostate-specific antigen,
and/or the estrogen receptor.
19. The method of claim 1, wherein said method is performed in an
aqueous medium.
20. The method of claim 1, wherein said method is performed in an
organic solvent.
21. A nanoparticle produced according to the methods of claim
1.
22. A composition comprising the nanoparticle of claim 21 and a
pharmaceutically acceptable carrier.
23. A method of treating a disease comprising the administration of
a nanoparticle according to claim 21 to a subject in an amount
sufficient to treat said disease.
24. The method of claim 23, wherein said disease is selected from
lung cancer, breast cancer, prostate cancer, head and neck cancer,
ovarian cancer, skin cancer, testicular cancer, pancreatic cancer,
esophageal cancer, colorectal cancer, kidney cancer, cervical
cancer, gastrointestinal cancer, viral infections, bacterial
infections or inflammation.
25. A polymer produced according to the method of claim 1, said
polymer comprising a targeting agent covalently bound to the
.alpha. terminus of a poly(ethylene glycol) polymer and at least
one second polymer covalently bound to said poly(ethylene glycol)
polymer via a functional group present on the .omega. terminus of
said poly(ethylene glycol).
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application 61/061,712, filed Jun. 16, 2008, the disclosure
of which is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] The delivery of a drug to a patient with controlled-release
of the active ingredient has been an active area of research for
decades and has been fueled by the many recent developments in
polymer science. In addition, controlled release polymer systems
can be designed to provide a drug level in the optimum range over a
longer period of time than other drug delivery methods, thus
increasing the efficacy of the drug and minimizing problems with
patient compliance.
[0003] Biodegradable particles have been developed as sustained
release vehicles used in the administration of small molecule
drugs, proteins and peptide drugs, and nucleic acids. The drugs are
typically encapsulated in a polymer matrix which is biodegradable
and biocompatible. As the polymer is degraded and/or as the drug
diffuses out of the polymer, the drug is released into the
body.
[0004] Targeting controlled release polymer systems (e.g., targeted
to a particular tissue or cell type or targeted to a specific
diseased tissue but not normal tissue) is desirable because it
reduces the amount of a drug present in tissues of the body that
are not targeted. This is particularly important when treating a
condition such as cancer where it is desirable that a cytotoxic
dose of the drug is delivered to target cells without killing the
surrounding tissue. Accordingly, a need exists to develop delivery
systems which can deliver therapeutic levels of drug to treat
diseases such as cancer, while also reducing patient side
effects.
BRIEF SUMMARY OF THE INVENTION
[0005] This application provides methods of making nanoparticles
using pre-functionalized poly(ethylene glycol) (also referred to as
PEG) as a macroinitiator for the synthesis of diblock copolymers.
These diblock copolymers comprise a functional PEG polymer block
bearing a targeting agent on one of its termini and a second
biocompatible and biodegradable hydrophobic polymer block (e.g. a
poly(ester)). The poly(ethylene glycol) is hetero-bifunctional with
a targeting agent (TA) covalently bound to its .alpha. terminus and
a polymerization initiating functional group (e.g., a hydroxyl
group) present on its .omega. terminus. Alternatively, the
poly(ethylene glycol) is functionalized with a TA on its .alpha.
terminus and a functional group capable of covalent attachment to a
poly(ester) that in turn is functionalized with a reactive end
group. Examples include an amino-terminated PEG and a carboxylic
acid terminated poly(ester) or an azide terminated PEG and an
alkyne terminated poly(ester). Nanoparticles produced according to
the disclosed methods and their use in the treatment of various
diseases and disorders is also provided.
BRIEF DESCRIPTION OF THE FIGURES
[0006] FIGS. 1A-C: Proton NMR spectra of HO-PEG-lys-urea-glu
(protected), lot numbers 11-189-1 and 11-176-1. FIG.
1A--HO-PEG-lys-urea-glu (protected) Lot# 11-176-1. 1H NMR spectrum
expansion #1 used for lys-urea-glu (protected) content calculation
(% lys-urea-glu (protected) end functionalization=[Int.
.delta.1.95-2.1]*2/[Int.
.delta.2.15-2.3]*100%=[2*10847/25777]*100=84%). FIG.
1B--HO-PEG-lys-urea-glu (protected) Lot# 11-176-1; 1H NMR spectrum
expansion #2. FIG. 1C--HO-PEG-lys-urea-glu (protected) Lot#
11-176-1; 1H NMR spectrum expansion #3.
[0007] FIGS. 2A-B: Size Exclusion Chromatograms (SEC) of
HO-PEG-lys-urea-glu (protected), lot numbers 11-189-1 (FIG. 2B) and
11-176-1 (FIG. 2A).
SEC Conditions:
[0008] Detector: Refractive Index detection (RI); RI Detector
temperature=35.degree. C. Columns: Water Styragel (HR1, 100-5000
Da; HR3, 500-30000 Da; HR4, 5000-600,000 Da in series).
Column Temperature=30.degree. C.;
[0009] Mobile phase: Chloroform; Flow rate: 1 mL/min Sample
concentration=10 mg/mL, Injection volume=50 uL (11-176-1) and 100
uL (11-189-1 coded 44-44-6 in chromatogram legends below); Number
and Weight Average Molecular Weight (M.sub.n and M.sub.w) and
Polydispersity (M.sub.w/M.sub.n) were obtained relative to narrow
disperse Poly(ethylene glycol) Standards (Polymer Standards Service
USA, Warwick, R.I.). A fourth order polynomial fit
(R.sup.2=0.99957; Standard Error=0.02) was used for calibration.
11-176-1 (FIG. 2A) and 11-189-1 (FIG. 2B) SEC Chromatograms
[0010] FIGS. 3A-F: Proton NMR spectra of PLA-PEG-lys-urea-glu
(protected), lot numbers 11-187-1, 11-188-1 and 11-198-1.
NMR Instrument: Bruker 400 MHz
[0011] NMR Experiment parameters: Solvent: CDCl.sub.3; Pulse Width:
7.5 usec; Pulse Delay: 5 sec; Number of scans: 128
[0012] FIG. 3A--PLA-PEG-lys-urea-glu (protected), Lot # 11-187-1;
1H NMR Spectrum Expansion #1 showing lactide methine and allyl end
group peak used in the determination the determination of the
absolute number average molar mass (Mn) of PLA-PEG-lys-urea-glu
(protected). Method for determination of absolute number average
molar mass (M.sub.n) of PLA-PEG-lys-urea-glu (protected):
M.sub.n(PLA-PEG-lys-urea-glu (protected)=M.sub.n(PEG-lys-urea-glu
(protected)+(Molar mass of lactide repeat unit*((Int. .delta.5.1
ppm-5.3 ppm)*3)/(Int..delta.5.85 ppm-5.95
ppm)=5000+((72*10,000)*3)/143)=20,105 Da. FIG.
3B--PLA-PEG-lys-urea-glu (protected), Lot# 11-187-1; 1H NMR
Spectrum Expansion #2 showing PEG peak and lactide methyl peaks of
PLA-PEG-lys-urea-glu (protected). FIG. 3C--PLA-PEG-lys-urea-glu
(protected), Lot# 11-188-1; 1H NMR Spectrum Expansion #1 showing
lactide methine and allyl end group peak used in the determination
the determination of the absolute number average molar mass (Mn) of
PLA-PEG-lys-urea-glu (protected). Method for determination of
absolute number average molar mass (M.sub.n) of
PLA-PEG-lys-urea-glu (protected): M.sub.n(PLA-PEG-lys-urea-glu
(protected)=M.sub.n(PEG-lys-urea-glu (protected)+(Molar mass of
lactide repeat unit*((Int. .delta.5.1 ppm-5.3
ppm)*3)/(Int..delta.5.85 ppm-5.95
ppm)=5000+((72*10,000)*3)/137)=20,766 Da. FIG.
3D--PLA-PEG-lys-urea-glu (protected), Lot# 11-188-1; 1H NMR
Spectrum Expansion #2 showing PEG peak and lactide methyl peaks of
PLA-PEG-lys-urea-glu (protected). FIG. 3E-PLA-PEG-lys-urea-glu
(protected), Lot# 11-198-1; 1H NMR Spectrum Expansion #1 showing
lactide methine and allyl end group peak used in the determination
the determination of the absolute number average molar mass (Mn) of
PLA-PEG-lys-urea-glu (protected). Method for determination of
absolute number average molar mass (M.sub.n) of
PLA-PEG-lys-urea-glu (protected): M.sub.n(PLA-PEG-lys-urea-glu
(protected)=M.sub.n(PEG-lys-urea-glu (protected)+(Molar mass of
lactide repeat unit*((Int. .delta.5.1 ppm-5.3
ppm)*3)/(Int..delta.5.85 ppm-5.95
ppm)=5000+((72*10,000)*3)/156)=18,846 Da. FIG.
3F-PLA-PEG-lys-urea-glu (protected), Lot# 11-198-1; 1H NMR Spectrum
Expansion #2 showing PEG peak and lactide methyl peaks of
PLA-PEG-lys-urea-glu (protected).
[0013] FIG. 4: Size Exclusion Chromatograms (SEC) of
HO-PEG-lys-urea-glu (protected), lot numbers 11-189-1 (labeled
44-44-6 for SEC analysis) and 11-176-1.
Size Exclusion Chromatographic Conditions:
[0014] Detector: Refractive Index detection (RI); RI Detector
temperature=35.degree. C. Columns: Water Styragel (HR1, 100-5000
Da; HR3, 500-30000 Da; HR4, 5000-600,000 Da in series). Column
Temperature=30.degree. C.; Mobile phase: Chloroform; Flow rate: 1
mL/min Sample concentration=10 mg/mL, Injection volume=20 uL;
Number and Weight Average Molecular Weight (M.sub.n and M.sub.w)
and polydispersity (M.sub.w/M.sub.n) were obtained relative to
narrow disperse Poly(styrene) Standards (Shodex Standards,
Kawasaki, Japan). A fourth order polynomial fit (R.sup.2=0.999927;
Standard Error=0.008) was used for calibration.
[0015] FIGS. 5A-E: Efficiency of deprotection and estimate of Molar
mass of crude PLA-PEG-lys-urea-glu (prior to palladium removal) by
.sup.1H NMR Spectroscopy.
NMR Instrument: Bruker 400 MHz
[0016] NMR Experiment parameters: Solvent: CDCl.sub.3; Pulse Width:
7.5 usec; Pulse Delay: 5 sec; Number of scans: 128
[0017] FIG. 5A--Crude PLA-PEG-lys-urea-glu Lot# 11-190-1; 1H NMR
Spectrum Expansion #1 showing: a) lactide methine peak, b) peaks of
aromatic protons of tetrakis(triphenyl-phosphine)palladium (0), and
c) absence of residual allyl peaks (.delta. 5.85-5.95) indicating
quantitative removal of protecting groups. FIG. 5B--Crude
PLA-PEG-lys-urea-glu Lot# 11-190-1; 1H NMR Spectrum Expansion #2
showing the PEG and lactide methyl peaks. FIG. 5C--Crude
PLA-PEG-lys-urea-glu Lot# 11-191-1; 1H NMR Spectrum Expansion #1
showing: a) lactide methine peak, b) peaks of aromatic protons of
tetrakis(triphenyl-phosphine)palladium (0), and c) <3% (relative
to the PLA-PEG-lys-urea-glu (protected) precursor) residual allyl
peaks (.delta. 5.85-5.95) indicating >96% removal of protecting
groups. FIG. 5D--Crude PLA-PEG-lys-urea-glu Lot# 11-191-1; 1H NMR
Spectrum Expansion #2 showing PEG and lactide methyl peaks. FIG.
5E--Crude PLA-PEG-lys-urea-glu Lot# 11-199-1; 1H NMR Spectrum
Expansion #1 showing: a) lactide methine peak, b) absence of
residual allyl peaks (.delta. 5.85-5.95) indicating quantitative
removal of protecting groups. FIG. 5F--Crude PLA-PEG-lys-urea-glu
Lot# 11-199-1; 1H NMR Spectrum Expansion #2 showing PEG and lactide
methyl peaks.
[0018] FIGS. 6A-B: Molar Mass and PEG fraction of purified
PLA-PEG-lys-urea-glu by .sup.1H NMR Spectroscopy.
NMR Instrument: Bruker 400 MHz
[0019] NMR Experiment parameters: Solvent: CDCl.sub.3; Pulse Width:
7.5 usec; Pulse Delay: 5 sec; Number of scans: 128
[0020] FIG. 6A--PLA-PEG-lys-urea-glu Lot# 44-49-1; 1H NMR Spectrum
Expansion #1 showing: a) lactide methine peak, b) absence of
residual allyl peaks (.delta. 5.85-5.95) indicating quantitative
removal of protecting groups. FIG. 6B--Purified
PLA-PEG-lys-urea-glu Lot# 44-49-1; 1H NMR Spectrum Expansion #2
showing PEG and lactide methyl peaks.
[0021] FIG. 7: Molecular weight of PLA-PEG-lys-urea-glu and the
protected precursor PLA-PEG-lys-urea-glu (protected) by Inherent
Viscosity measurements.
[0022] FIG. 8: Palladium Content in crude (prior to palladium
removal) PLA-PEG-lys-urea-glu lot number 44-48-1 (sample code
11-204-1) and purified (after palladium removal)
PLA-PEG-lys-urea-glu lot number 44-49-1 (sample code 11-204-2)
determined by ICP Spectrometry.
DETAILED DISCLOSURE OF THE INVENTION
[0023] This application provides methods of making nanoparticles
using pre-functionalized poly(ethylene glycol) (also referred to as
PEG) as a macroinitiator for the synthesis of diblock copolymers.
These diblock copolymers comprise a bio-active poly(ethylene
glycol) block and a second biocompatible and biodegradable
hydrophobic polymer block (e.g. a poly(ester)). The poly(ethylene
glycol) is hetero-bifunctional with a targeting moiety (agent)
covalently bound to its .alpha. terminus and a polymerization
initiating functional group (e.g., a hydroxyl group) present on its
.omega. terminus.
[0024] The subject application also provides for the use of
derivatives of targeting agents (TA's) (e.g., analogs where
functional groups, such as carboxylic acids or other functional
groups, are protected) in the synthesis of the poly(ethylene
glycol) polymer to which TA's are attached that improve its
solubility and avoid potential side reactions. After a protected
targeting agent (PRO-TA) is coupled to the PEG polymer at the
.alpha. terminus, the functionalized PEG (HO-PEG-TA-PRO) is
utilized as a macroinitiator to synthesize a poly(ester) block. For
example, HO-PEG-TA-PRO is added to a mixture of cyclic lactone
monomers such as lactide, glycolide or caprolactone and the mixture
is heated to melt conditions. A polymerization catalyst, such as
tin (II) 2-ethylhexanoate is then added to the monomer/initiator
melt. The resulting polymer is purified from un-reacted monomer and
polymerization catalyst by precipitation into a non-solvent mixture
such as ether/hexane (70/30) and recovered by decantation followed
by vacuum drying. Subsequent deprotection of the protected
functional groups can be performed to regain the original
chemically active functional group (e.g., a carboxylic acid). This
approach enables the desired polymerization reaction to proceed
efficiently and avoids side reactions between the polymerization
catalyst and the functional groups on the targeting moiety in its
native unprotected form. Such side reactions result in the
formation of cross-linked insoluble gels rather than the desired
linear poly(ester) polymer. In addition, the composition and
molecular weight of the poly(ester) can be tuned as desired by
controlling the composition of the monomer melt and the molar ratio
of the HO-PEG-TA(PRO) to the monomers. Furthermore, the protected
targeting moiety is freely soluble in a wide range of organic
solvents relative to the un-protected analogues. This enables the
coupling of the targeting moiety to poly(ethylene glycol) with a
high degree of polymer end group functionalization. Non-limiting
examples of protecting groups for various targeting agent
functional groups, as disclosed herein, are:
TABLE-US-00001 Functional group Protecting group Alcohol Alkyl
allyl carbonate, Alloc-OR Alcohol Methyl ether Alcohol Methyl ether
Alcohol Methyl ether Alcohol Allyl benzylcarbonate, ROCO.sub.2Bn
Phenol Allyl carbonate Phenol Methyl ether Phenol Benzyloxymethyl
ether - BOM Phenol Methoxyethoxymethyl Ether - MEM ether Thiol
S-p-Methoxybenzyl thioether Thiol S-Triphenylmethyl thioether
(Tr-SR) Thiol 4-methoxytrityl (Mtt-SR) Thiol S-t-butyl thioether
Thiol S-[Tricarbonyl[1,2,3,4,5,.eta.]-2,4-dicyclohexadiene-1
-yl]-iron(1+) thioether Amine N-allylamine Amine N-benzylamine
Amine Allyl carbamate (Alloc-NR.sub.2) Amine Benzyl
carbamate-benzyloxycarbonyl (CBz) Amine p-methoxybenzyl carbamate -
Moz Amine p-nitrobenzyl carbamate - PNZ Carboxylic acid Allyl ester
Carboxylic acid Propargyl ester Carboxylic acid Benzyl ester -
RCO.sub.2Bn
[0025] Additionally, this application provides a method for
efficient coupling of the un-protected targeting moiety to
poly(ethylene glycol) under aqueous conditions. In this approach,
the pH of the reaction medium is used to maximize yield of the
desired product. Advantages to this aspect of the invention relate
to the utilization of the high water solubility of the targeting
agent (TA). For example, the acid end group of a
.alpha.-azide-.omega.-carboxylic acid poly(ethylene glycol)
(N.sub.3-PEG-CO.sub.2H) is first activated by conversion to the
succinimide ester N.sub.3-PEG-COSu) by reaction with
N-hydroxysuccinimide (NHS) and ethyl
dimethylaminopropylcarbodiimide hydrochloride (EDC) or
dicyclohexylcarbodiimide (DCC) under anhydrous organic solvent
conditions such as in dichloromethane. The activated poly(ethylene
glycol) (N.sub.3-PEG-COSu) is subsequently purified from small
molecule reagents precipitation into an anhydrous non-solvent such
as ether-hexane (70/30). The un-protected form of the
amine-functional targeting agent (H.sub.2N-TA) is dissolved in
bicarbonate buffer (pH=9.7) and pH then lowered to 7.4 using
aqueous sodium hydroxide. The activated acid (N.sub.3-PEG-COSu) is
dissolved in DI water is added dropwise to a large molar excess of
the H.sub.2N-TA solution. The succinimide ester of N.sub.3-PEG-COSu
is stable at pH=5.5-6, while the amide bond forming reaction with
N.sub.2N-TA is efficient under pH=7.4 conditions. This strategy
exposes the N.sub.3-PEG-COSu to pH=7.4 only when a large excess of
amine-functional targeting moiety is also present. Thus, conversion
to the desired PEG-targeting agent conjugate (N.sub.3-PEG-TA) while
minimizing exposure of the N.sub.3-PEG-COSu to basic conditions,
thereby avoiding hydrolysis of the succinimide ester. The
N.sub.3-PEG-TA is subsequently coupled to an alkyne terminal
poly(ester) using copper catalyst under conventional click
chemistry conditions in an organic solvent such as
dimethylformamide (DMF) or dimethylsulfoxide (DMSO). Alkyne
terminal poly(ester) is prepared by ring opening polymerization of
lactide and glycolide monomers using for example propargyl alcohol
as polymerization initiator and tin (II) 2-ethyl hexanoate as
polymerization catalyst.
[0026] As an example, a targeting moiety/agent may target or cause
the particle to become localized at specific locations within a
subject and the bioactive agent/therapeutic agent is thus delivered
to a target site. In one embodiment, a drug or therapeutic agent
can be released in a controlled release manner from the particle
and allowed to interact locally with the particular targeted site
(e.g., a tumor). The term "controlled release" (and variations of
that phrase (e.g., in the context of "controlled-release system"))
is generally meant to encompass release of a therapeutic agent
(e.g., a drug) at a selected site in a controllable rate, interval,
and/or amount. "Controlled release" encompasses, but is not
necessarily limited to, substantially continuous delivery,
patterned delivery (e.g., intermittent delivery over a period of
time that is interrupted by regular or irregular time intervals) or
delivery of a bolus of a selected therapeutic agent (or various
combinations thereof) as a predetermined, discrete amount over a
relatively short period of time (e.g., a few seconds or
minutes).
[0027] Examples of second biocompatible and biodegradable
hydrophobic polymer blocks that can be used in the manufacture of
the claimed nanoparticles can be polyesters. Examplary polyesters
suitable for use in the manufacture of the disclosed nanoparticles
include copolymers comprising lactic acid and glycolic acid units,
such as polylactic acid-co-glycolic acid) and
poly(lactide-co-glycolide), collectively referred to herein as
"PLGA"; and homopolymers comprising glycolic acid units, referred
to herein as "PGA," and lactic acid units, such as poly-L-lactic
acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide,
poly-D-lactide, and poly-D,L-lactide, collectively referred to
herein as "PLA." In some embodiments, exemplary polyesters include,
for example, polyhydroxyacids; PEGylated polymers and copolymers of
lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA,
PEGylated PLGA, and derivatives thereof. In some embodiments,
polyesters include, for example, polyanhydrides, poly(ortho ester)
PEGylated poly(ortho ester), poly(caprolactone), PEGylated
poly(caprolactone), poly(L-lactide-co-L-lysine), poly(serine
ester), poly(4-hydroxy-L-proline ester),
poly[a-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0028] Second biocompatible and biodegradable hydrophobic polymer
block (e.g., polyesters) utilized in the manufacture of the
disclosed nanoparticles can contain functional groups that react
with the .omega. terminus of the poly(ethylene glycol).
Non-limiting examples of such functional groups include, and are
not limited to, amines, hydroxyl groups, carboxylic acid groups,
NHS groups, alkyne groups or azide groups. Groups with which the
second biocompatible and biodegradable hydrophobic polymer block
functional groups react at the w terminus of the poly(ethylene
glycol) polymer are provided in the following table.
TABLE-US-00002 Reactive group at the .omega. terminus of Polyester
functional group poly(ethylene glycol) N-hydroxysuccinimide (NHS)
Amine Amine Hydroxyl, Carboxylic acid Hydroxyl Amine Carboxylic
acid Amine Alkyne Azide
[0029] Targeting agents disclosed herein can contain, or be
modified to contain, a functional group that can be reacted with
the .alpha. terminus of a polymer (e.g., PEG) in order to produce a
polymer conjugated to a targeting moiety. The functional groups
include any moiety that can be used to create a covalent bond with
a polymer (e.g., PEG), such as amino, hydroxy, azide, alkyne and
thio. For example, targeting agents can be can be substituted with
NH.sub.2, SH or OH, which are either bound directly to the
targeting agent or via an additional group, e.g., alkyl or phenyl.
In a non-limiting example, aniline, alkyl-NH.sub.2 (e.g.,
(CH.sub.2).sub.1-6NH.sub.2), or alkyl-SH (e.g.,
(CH.sub.2).sub.1-6NH.sub.2) can be used to link the targeting agent
to a polymer via the free NH.sub.2 and SH groups to form a covalent
bond.
[0030] The conjugation of a functionalized PEG polymer (a PEG
polymer comprising one or more targeting agents at its .alpha.
terminus and reactive functional groups at the .omega. terminus)
and a second biocompatible and biodegradable hydrophobic polymer
can be performed according to methods known in the art via
functional groups at the .omega. terminus of a functionalized PEG
polymer and reactive groups present in the second biocompatible and
biodegradable hydrophobic polymer. For example, EDC-NHS chemistry
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and
N-hydroxysuccinimide) or a reaction involving a maleimide or a
carboxylic acid can be used. The conjugation of a poly(ester) and a
poly(ether) to form a poly(ester-ether), can be performed in an
organic solvent, such as, but not limited to, dichloromethane,
acetonitrile, chloroform, dimethylformamide, tetrahydrofuran,
acetone, or the like. Specific reaction conditions can be
determined by those of ordinary skill in the art using no more than
routine experimentation.
[0031] The conjugation of a functionalized PEG polymer (a PEG
polymer comprising one or more targeting agents (TA) at its .alpha.
terminus and reactive functional groups at the .omega. terminus)
and a second biocompatible and biodegradable hydrophobic polymer
can also be performed using chemistries that are orthogonal to the
amide bond forming EDC-NHS chemistry. Such methods include "click"
chemistry techniques. For example, an alkyne terminated poly(ester)
may be reacted with a heterobifunctional polyethylene glycol)
bearing a TA at its .alpha.-terminus and an alkyne reactive azide
moiety at its .omega.-terminus. The conjugation of a poly(ester)
and a poly(ether) to form a poly(ester-ether), can be performed in
an organic solvent, such as, but not limited to, dichloromethane,
acetonitrile, chloroform, dimethylformamide, tetrahydrofuran,
acetone, or the like. Conventional "click" chemistry catalysts such
as copper sulfate may be employed.
[0032] In another set of embodiments, a conjugation reaction may be
performed by reacting a polymer that comprises a carboxylic acid
functional group (e.g., a poly(ester-ether) compound) with a
polymer or other moiety (such as a targeting moiety) comprising an
amine. For instance, a targeting moiety, such as a low-molecular
weight PSMA ligand, may be reacted with an amine to form an
amine-containing moiety, which can then be conjugated to the
carboxylic acid of the polymer. Such a reaction may occur as a
single-step reaction, i.e., the conjugation is performed without
using intermediates such as N-hydroxysuccinimide or a maleimide.
The conjugation reaction between the amine-containing moiety and
the carboxylic acid-terminated polymer (such as a poly(ester-ether)
compound) may be achieved, in one set of embodiments, by adding the
amine-containing moiety, solubilized in an organic solvent such as
(but not limited to) dichloromethane, acetonitrile, chloroform,
tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines,
dioxane, or dimethysulfoxide, to a solution containing the
carboxylic acid-terminated polymer. The carboxylic acid-terminated
polymer may be contained within an organic solvent such as, but not
limited to, dichloromethane, acetonitrile, chloroform,
dimethylformamide, tetrahydrofuran, or acetone. Reaction between
the amine-containing moiety and the carboxylic acid-terminated
polymer may occur spontaneously, in some cases. Unconjugated
reactants may be washed away after such reactions, and the polymer
may be precipitated in solvents such as, for instance, ethyl ether,
hexane, methanol, or ethanol.
[0033] Another aspect of the invention is directed to particles
that include polymer conjugates such as the ones described above.
The particles may have a substantially spherical (i.e., the
particles generally appear to be spherical), or non-spherical
configuration. For instance, the particles, upon swelling or
shrinkage, may adopt a non-spherical configuration. In some cases,
the particles may include polymeric blends. For instance, a polymer
blend may be formed that includes a first PEG polymer comprising a
targeting moiety (i.e., a low-molecular weight PSMA ligand) and a
second polymer comprising a biocompatible polymer (e.g., lacking a
targeting moiety). By controlling the ratio of the first and second
polymers in the final polymer, the concentration and location of
targeting moiety in the final polymer may be readily controlled to
any suitable degree.
[0034] As discussed above, the polymer may be PLGA in some
embodiments. PLGA is a biocompatible and biodegradable co-polymer
of lactic acid and glycolic acid, and various forms of PLGA are
characterized by the ratio of lactic acid:glycolic acid. Lactic
acid can be L-lactic acid, D-lactic acid, or D,L-lactic acid. The
degradation rate of PLGA can be adjusted by altering the lactic
acid-glycolic acid ratio. In some embodiments, PLGA to be used in
accordance with the present invention is characterized by a lactic
acid:glycolic acid ratio of approximately 85:15, approximately
75:25, approximately 60:40, approximately 50:50, approximately
40:60, approximately 25:75, or approximately 15:85.
[0035] In particular embodiments, by optimizing the ratio of lactic
acid to glycolic acid monomers in the polymer of the nanoparticle
(e.g., the PLGA block copolymer or PLGA-PEG block copolymer),
nanoparticle parameters such as water uptake, therapeutic agent
release (e.g., "controlled release") and polymer degradation
kinetics can be optimized. Yet other embodiments provide polymers
that may be one or more acrylic polymers. In certain embodiments,
acrylic polymers include, for example, copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, aminoalkyl methacrylate copolymer, methacrylic acid
alkylamide copolymer, poly(methyl methacrylate), aminoalkyl
methacrylate copolymer, glycidyl methacrylate copolymers,
polycyanoacrylates, and combinations comprising one or more of the
foregoing polymers.
[0036] In some other embodiments, polymers can be cationic
polymers. In general, cationic polymers are able to condense and/or
protect negatively charged strands of nucleic acids (e.g. DNA, RNA,
or derivatives thereof). Amine-containing polymers such as
poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and
Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethylene
imine) (PEI; Boussif et al, 1995, Proc. Natl. Acad. Sci., USA,
1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo
et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al.,
1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,
Bioconjugate Chem., 4:372) are positively-charged at physiological
pH, form ion pairs with nucleic acids, and mediate transfection in
a variety of cell lines.
[0037] In yet other embodiments, polymers can be degradable
polyesters bearing cationic side chains (Putnam et al., 1999,
Macromolecules, 32:3658; Barrera et al., 1993, J. Am. Chem. Soc.,
115:11010; Urn et al., 1999, J. Am. Chem., Soc., 121:5633; and Zhou
et al, 1990, Macromolecules, 23:3399). Incorporation of hydrophobic
comonomers (e.g., lactide, glycolide, caprolactone, or hydrophobic
amino acids such as alanine, valine, leucine, isoleucine or
phenylalanine) into the polymer backbone to an extent that will
impart hydrophobic character to the copolymer enables formation of
nanoparticles with a degree of cationic character within the
particle core. This in turn enables encapsulation of drugs such as
siRNA into the nanoparticle core. Examples of these polyesters
include poly(L-lactide-co-L-lysine) (Barrera et al, 1993, J. Am.
Chem. Soc., 115:11010), poly(serine ester) (Zhou et al, 1990,
Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam
et al, 1999, Macromolecules, 32:3658; and Lim et al, 1999, J. Am.
Chem. Soc., 121:5633). Poly(4-hydroxy-L-proline ester) was
demonstrated to condense plasmid DNA through electrostatic
interactions, and to mediate gene transfer (Putnam et al, 1999,
Macromolecules, 32:3658; and Lim et al, 1999, J. Am. Chem. Soc.,
121:5633). These new polymers are less toxic than poly(lysine) and
PEI, and they degrade into non-toxic metabolites.
[0038] In a particular embodiment, the molecular weight of the
polymers of the nanoparticles of the invention are optimized for
effective treatment of cancer, e.g., prostate cancer. For example,
the molecular weight of the polymer influences nanoparticle
degradation rate (particularly when the molecular weight of a
biodegradable polymer is adjusted), solubility, water uptake, and
drug release kinetics (e.g. "controlled release"). As a further
example, the molecular weight of the polymer can be adjusted such
that the nanoparticle biodegrades in the subject being treated
within a reasonable period of time (ranging from a few hours to 1-2
weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks, etc.). In particular
embodiments of a nanoparticle comprising a copolymer of PEG and
PLGA, the PEG has a molecular weight of 1,000-20,000 Da (e.g.,
5,000-20,000, e.g., 10,000-20,000) and, in some embodiments, 5000
Da, and the PLGA has a molecular weight of 5,000-100,000 Da (e.g.,
20,000-70,000, e.g., 20,000-50,000), or in some embodiments,
15,000-30,000 Da.
[0039] The nanoparticles disclosed herein can be used for the
treatment of various diseases and disorders within a subject. A
subject may be a human or non-human animal. Examples of subjects
include, but are not limited to, a mammal such as a dog, a cat, a
horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat, a rat, a
mouse, a guinea pig, a hamster, a primate, a human or the like.
[0040] The subject invention provides a number of methods of making
nanoparticles comprising one or more targeting agent and/or one or
more bioactive moiety/therapeutic agent. In various aspects of the
invention, a functionalized targeting agent conjugated diblock
copolymer is synthesized by initiating the polymerization of
biocompatible and biodegradable hydrophobic polymer (such as
poly(ester)) from the .omega. terminus of the poly(ethylene
glycol). Scheme 1 illustrates this process as exemplified by a
poly(ethylene glycol) bearing a hydroxyl functional group on its
.alpha.-terminus and targeting agent (TA) on its co-terminus.
Polymerization of a poly(ester) block from this hydroxyl terminus
yields the desired diblock copolymer bearing a TA covalently bound
to its PEG terminus.
##STR00001##
[0041] Alternatively, a diblock copolymer bearing a targeting agent
(TA) on its poly(ethylene glycol) terminus may be prepared by
conjugation of a suitably protected form of the targeting agent
(TA-PRO) to the poly(ethylene glycol) polymer (yielding
HO-PEG-TA(PRO)), and subsequently using this macroinitiator in the
ring opening polymerization of a cyclic lactone monomer such as
lactide, glycolide or a mixture thereof. Removal of the protecting
groups of the targeting agent using standard deprotection
methodology provides the desired Poly(ester)-block-PEG-TA (see
scheme 2).
[0042] Alternatively, a functional diblock copolymer
(Poly(ester)-PEG-TA) may be prepared by the covalent coupling of
the targeting PEG-TA to a pre-formed poly(ester). For example, a
carboxylic acid terminated poly(lactide-co-glycolide)
(Poly(ester)-CO.sub.2H) and poly(ethylene glycol) bearing the
targeting agent on its .alpha.-terminus and an acid reactive amino
functional group on its co-terminus (H.sub.2N-PEG-TA) may be
reacted under organic solvent conditions.
##STR00002##
[0043] Targeting agents (TA's) may be comprised of individual
natural or non-natural amino acids, or a combination of two or more
amino acid residues covalently bound either by a amide or a urea
linkage. TA's may also be comprised of nucleic acids. As such, TA's
may contain side chain moieties bearing functional groups including
carboxylic acids, amines, thiols, alcohols, phenols, guanidine,
purines (such as adenine, guanine), pyrimidines (such as cytosine,
uracil, thymine). The method of scheme 2 is preferred over the
method of scheme 1 when TA's contain functional groups that may
either initiate a ring opening polymerization (such as an alcohol,
a phenol, an amine, or a thiol) or react with and structurally
alter the polymerization catalyst (such as guanidine,
1,5-pentandioic acid moieties as found when a dipeptide or
polypeptide TA has a glutamic acid residue on its C-terminus, or a
urea linkage capable of binding to the polymerization
catalyst).
[0044] This invention also describes methods for the synthesis of
covalent conjugates of heterobifunctional polyethylene glycol) and
targeting agents (TA's). Poly(ethyleneglycol) (PEG) bearing a
hydroxyl group at its .alpha.-terminus and a reactive functional
group on its co-terminus such as a carboxylic acid, an aldehyde, an
azide, an alkyne, a maleimido group, are described herein. Such
heterobinfunctional PEG's may be reacted with a TA bearing an
amine, a thiol, an alkyne or an azide moiety to yield a covalent
conjugate of PEG and TA (HO-PEG-TA). Preferred reactive moieties
include amine and carboxylic acid and amine and aldehyde that yield
naturally occurring linkages such as amides or secondary amines.
Scheme 3 provides chemical synthetic methodologies to enable such
covalent conjugation.
##STR00003##
[0045] This invention also describes methods for the synthesis of
HO-PEG-TA(PRO) conjugates under anhydrous organic solvent condition
using the preferred amidation reaction of an amine moiety of the
TA(PRO) and an acid terminus of PEG. Scheme 4 illustrates the
method used for covalent conjugation of HO-PEG-CO.sub.2H to a allyl
protected lysine-urea-glutamic acid (lys-urea-glu) targeting agent.
Use of the allyl protected analog of the lys-urea-glu targeting
agent improves its solubility in organic solvents most suitable
(such as dichloromethane) for EDC/NHS acid activation chemistry.
Amidation reaction under anhydrous organic solvent conditions
enables a high degree of conjugation efficiency and yields over 80%
end group functionalization in HO-PEG-lys-urea-glu(allyl
protected). Furthermore, the allyl protected lys-urea-glu (TA(PRO))
enables the ring opening polymerization of lactone monomers without
undesirable side reaction with the preferred polymerization
catalyst (tin (II) 2-ethylhexanoate).
##STR00004##
[0046] This invention also describes methods for the synthesis of
HO-PEG-TA(PRO) conjugates under organic solvent condition using
reductive alkylation reaction of an amine moiety of the TA(PRO) and
an aldehyde terminus of PEG. Scheme 5 illustrates the method used
for covalent conjugation of HO-PEG-CHO to allyl protected
lysine-urea-glutamic acid (TA(PRO)). Use of the allyl protected
analog of the lys-urea-glu targeting moiety improves its solubility
in organic solvents (such as dichloromethane, dimethylformamide)
suitable for reductive alkylation chemistry. Reductive alkylation
under organic solvent conditions enables a high degree of
conjugation efficiency and yields over 80% end group
functionalization in HO-PEG-lys-urea-glu(allyl protected).
##STR00005##
This invention also describes the ring opening polymerization of
cyclic lactone monomers using the HO-PEG-lys(urea)glu(allyl
protected) (HO-PEG-TA(PRO)) conjugate as a macroinitiator and
tin(II) 2-ethyl hexanoate as polymerization catalyst under monomer
melt conditions at 130.degree. C. (scheme 6), this method is also
referred to as the "polymerization from" approach. For example,
poly(D,L-lactide)-block-poly(ethylene glycol)-lys-urea-glu(allyl
protected) obtained by such polymerization is then converted to
poly(D,L-lactide)-block-poly(ethylene glycol)-lys-urea-glu by
removal of the allyl protecting groups using organic base
(morpholine) and tetrakis(triphenylphosphine) palladium (0) as
catalyst (scheme 7). The deprotection reaction conditions are
optimized with regards to molar equivalents of morpholine,
palladium catalyst and reaction time to enable quantitative removal
of allyl protecting groups (>98%, determined by NMR
spectroscopy), without any measurable reduction in the molar mass
(as determined by size exclusion chromatography and dilute solution
viscometry) of the block copolymer.
##STR00006##
This application also describes the removal of residual palladium
from the functionalized diblock copolymer PLA-PEG-lys-urea-glu
(PLA-PEG-TA). Scheme 8 shows the chemical structures of several
commercially available resins used for scavenging palladium
contaminants. Trimercaptotriazide (TMT) functional resin is
preferred for the removal of palladium from PLA-PEG-lys-urea-glu
samples with minimal loss of polymer yield in the palladium removal
step. Yield loss is observed when resins functionalized with
palladium binding moieties other than TMT are used. This is due to
interaction between the resin bound palladium binding moieties and
the TA in PLA-PEG-TA.
##STR00007##
This application also describes the covalent conjugation of
targeting agents (TA's) in their native unprotected form under
aqueous solution conditions to heterobifunctional poly(ethylene
glycol). This method utilizes the high water solubility of the
targeting moiety such as lys-urea-glu or other peptide based
targeting ligands. The solubility properties of the
PEG-lys-urea-glu conjugate are dominated by the poly(ethylene
glycol) polymer. Thus, unlike lys-urea-glu, PEG-lys-urea-glu is
soluble in common organic solvents including but not limited to
dichloromethane, dimethyl formamide, tetrahydrofuran, chloroform,
or dimethylsulfoxide. This enables covalent coupling of the
PEG-lys-urea-glu conjugate to end functional poly(ester) to yield
the desired Poly(ester)-PEG-lys-urea-glu (Poly(ester)-PEG-TA) under
organic solvent conditions where lys-urea-glu prior to the PEG
conjugation is insoluble or sparingly soluble. This approach is
referred to as the "coupling to" approach. Complete dissolution of
the end functional poly(ester) and the PEG-lys-urea-glu in a common
solvent is critical to obtaining a high yield of the desired
Poly(ester)-PEG-TA functional block copolymer.
##STR00008##
[0047] The PEG-lys-urea-glu is covalently coupled to an end
functional poly(ester) using chemistries that proceed without side
reactions with the carboxylic acid moieties of lys-urea-glu. For
example, an .alpha.-azido-.omega.-carboxylic acid may be conjugated
to the amino moiety of the lys-urea-glu by its reaction with the
carboxy terminus of such PEG.
[0048] The targeting agent functional PEG,
.alpha.-azido-.omega.-(lys-urea-glu)polyethylene glycol may be
prepared by synthesis of a heterobifunctional precursor such as
.alpha.-azido-.omega.-carboxy polyethylene glycol
(N.sub.3-PEG-CO.sub.2H) from commercially available starting
materials such as .alpha.-amino-.omega.-carboxy-poly(ethylene
glycol) and for example 4-azidophenyl isothiocyanate,
O-(2-azidoethyl)-O-[2-diglycolyl-amino)ethyl]heptaethylene glycol
or azide-PEG4-NHS using standard conjugation methodologies (scheme
9). The heterobifunctional polymer, N.sub.3-PEG-CO.sub.2H is
subsequently reacted with the amine functionality of lys-urea-glu
under aqueous conditions using methods illustrated in scheme
10.
##STR00009##
Alternatively, N.sub.3-PEG-NHS may be prepared by activation of the
carboxy terminus of N.sub.3-PEG-CO.sub.2H under anhydrous organic
solvent conditions such as in dichloromethane using organic soluble
ethyldimethyl aminopropylcarbodiimide (EDC), N-hydroxysuccinimide
(NHS) and diisopropyl ethylamine (DIEA) and subsequently purified
by precipitation into anhydrous ether/hexane (70/30). The activated
polymer (N.sub.3-PEG-NHS) is then recovered by filtration and
vacuum drying and stored in a dry environment under dry nitrogen (1
ppm water dry glove box for 48 hours) prior to storage at
-20.degree. C. N.sub.3-PEG-NHS prepared by this method is then used
to prepare N.sub.3-PEG-lys-urea-glu as outlined in steps 6 though
10 of scheme 10.
##STR00010##
[0049] As stated above, this application also describes the
covalent coupling of N.sub.3-PEG-lys-urea-glu to an end functional
poly(ester) bearing a alkyne moiety at its .alpha.-terminus and a
hydroxy terminus at its .omega.-terminus using well established
"click" chemistry techniques such as using a copper sulfate
catalyst under organic solvent conditions, for example in
dimethylformamide (DMF) (scheme 11). This "click" chemistry
methodology is particularly useful since the carboxylic acid
functionalities of lys-urea-glu are not reactive towards the alkyne
functionality of the poly(ester). Such orthogonal chemistry rules
out possible side reactions and permits the use long reaction times
between the poly(ester)-alkyne and N.sub.3-PEG-lys-urea-glu.
Furthermore, unlike amidation chemistry using for example
Poly(ester)-NHS, "click" chemistry is not moisture sensitive, thus
need for anhydrous conditions is not critical to ensure an
acceptable degree of covalent conjugation that provides >80% end
functionalization.
##STR00011##
The "polymerization from" approach of scheme 6 and 7 has the
following advantages: [0050] 1. Use of HO-PEG-TA(PRO) in the ring
opening polymerization ensures no cross-reactivity between the
functional groups of TA polymerization catalyst or monomer thereby
avoiding loss of control over the polymerization and contaminating
byproducts. [0051] 2. Absence of side reactions allows excellent
control of the molecular weight and composition of the
Poly(ester)-PEG-TA(PRO) [0052] 3. Adjustment of the composition of
the monomer feed and the molar ratio of the initiator to monomer
enables synthesis of the poly(ester) block of a wide range of
desired compositions and molecular weights. The ratio or the PEG to
the poly(ester) may also be varied at with ease. [0053] 4. The
PLA-PEG-TA(PRO) is soluble in common dueterated organic solvents
used for NMR spectroscopy such as chloroform-d, DMSO-d6, DMF-d7, or
THF-d4. This enables NMR analysis of Poly(ester)-PEG-TA(PRO)
providing quantitative information regarding critical polymer
characteristics such as extent of TA(PRO) end group on
Poly(ester)-PEG-TA(PRO) as well as absolute number average
molecular weight (M.sub.n). Provided relative molar mass
(determined by for example size exclusion chromatography or
inherent viscosity) of Poly(ester)-PEG-TA is equivalent to that of
its protected precursor (Poly(ester)-PEG-TA(PRO)), the absolute
M.sub.n of Poly(ester)-PEG-TA(PRO) (determined by NMR spectroscopy)
may be considered equal to that of its precursor. This enables
precise determination of polymer characteristic. [0054] 5. Precise
determination of Poly(ester)-PEG-TA characteristic such as its
absolute M.sub.n and the extent of TA end group functionalization
are critical for controlling the targeting properties of the
nanoparticles made from Poly(ester)-PEG-TA. Such nanoparticles are
prepared by blending known quantities of this functional polymer
(Poly(ester)-PEG-TA) with non-functional polymer (Poly(ester)-PEG)
to control the degree of targeting agent within the therapeutic
nanoparticle. The "polymerization from" approach of scheme 6 and 7
has the following shortcomings: [0055] 1. It requires the
functional groups of the TA to be protected. This may require use
of expensive and/or difficult to obtain starting materials when TA
is based on 2 or more amino acid residues or nucleic acids
containing side chain moieties that may interfere with the ring
opening polymerization. [0056] 2. It necessitates removal of the
protecting groups of TA(PRO) to yield the desired
Poly(ester)-PEG-TA. The deprotection reaction may require use of a
heavy metal catalyst that needs to be removed from the final
product. This adding a purification step to the process that may in
some cases be difficult to achieve with sufficient efficiency so as
to provide a polymeric material of pharmaceutically acceptable
purity. The "coupling to" approach has the following advantages:
[0057] 1. It does not require potentially expensive protected
analogues of the targeting agent (TA(PRO)'s) [0058] 2. It does not
require the deprotection of the TA(PRO) to obtain the desired
Poly(ester)-PEG-TA polymer thereby avoiding associated shortcomings
as described previously. The "coupling to" approach has the
following shortcomings: [0059] 1. It relies on coupling pre-made
end functional poly(ester)s to PEG-TA, hence control over the
composition and molecular weight of the poly(ester) requires
synthesis of poly(ester)s of various desired properties in advance
of the coupling reaction. [0060] 2. Coupling of macromolecular
entities such as an end functional Poly(ester) and PEG-TA is slow
due to poor diffusion characteristic of polymers and requires long
reaction times at high reagents concentrations to ensure acceptable
coupling efficiencies (>80%). [0061] 3. The method yields
Poly(ester)-PEG-TA. Diblock copolymers bearing TA's as an end group
are difficult to analyze by NMR spectroscopy due to differences in
solubility characteristics of the poly(ester)-PEG polymer and the
TA. Thus, quantitative analysis of end group functionality and
absolute M.sub.n is difficult using conventional methods such NMR
spectroscopy. In view of the advantages and shortcoming of the
"polymerization from" and "coupling to" approaches, the latter is
preferred over the former only when the TA(PRO) is expensive and/or
difficult to obtain or the deprotection conditions are difficult or
irreversibly contaminate the final polymeric product. Otherwise,
the "polymerization from" approach is preferred due to easy of
access to a large variety of polymer architectures and compositions
as well as ease of analytical characterization of the
Poly(ester)-PEG-TA(PRO) and hence the final product
(Poly(ester)-PEG-TA) in cases where equivalence of polymer
architecture may be established by other non-absolute yet
dependable analytical methods. The self-assembly of the
poly(ester)-PEG-X diblock copolymers in aqueous medium yields
nanoparticles comprising a hydrophobic core and a hydrophilic
poly(ethylene glycol) corona (exterior). The hydrophobic core bears
the bioactive/therapeutic agent while the hydrophilic corona
presents the targeting agent on the nanoparticle surface and
enables the nanoparticles to selectively bind to target sites
(e.g., diseased tissue) via interaction of the targeting agent with
a corresponding agent expressed at the target site.
[0062] Chemical Structures of abbreviations used:
##STR00012## ##STR00013##
Bioactive Moieties/Therapeutic Agents
[0063] As discussed above, therapeutic agents (bioactive moieties)
can be incorporated into the hydrophobic nanoparticle core in
aqueous medium or organic solvents and the nanoparticles can be,
subsequently, purified. Bioactive agents (therapeutic agents)
include, and are not limited to, therapeutic agents (e.g.
anti-cancer agents), diagnostic agents (e.g. contrast agents;
radionuclides; and fluorescent, luminescent, and magnetic
moieties), prophylactic agents (e.g. vaccines), and/or
nutraceutical agents (e.g. vitamins, minerals, etc.). As discussed
herein, bioactive agents may be administered to an individual as
disclosed herein. Exemplary therapeutic agents to be delivered in
accordance with the present invention include, but are not limited
to, small molecules (e.g. cytotoxic agents), nucleic acids (e.g.,
siRNA, RNAi, and mircoRNA agents), proteins (e.g. antibodies),
peptides, lipids, carbohydrates, hormones, metals, radioactive
elements and compounds, drugs, vaccines, immunological agents,
etc., and/or combinations thereof. In some embodiments, the agent
to be delivered is an agent useful in the treatment of cancer
(e.g., prostate cancer).
[0064] Some examples of therapeutic agents that can be delivered
within the nanoparticles produced in accordance with the disclosed
methods include, but are not limited to agents such as penicillins,
aminopenicillins, penicillins in conjunction with penicillinase
inhibitor and/or anti-fungal agents), cephalosporins, cephamycins
and carbapenems, fluoroquinolones, tetracyclines, macrolides and
aminoglycosides. Specific examples include, but are not limited to,
erythromycin, bacitracin zinc, polymyxin, polymyxin B sulfates,
neomycin, gentamycin, tobramycin, gramicidin, ciprofloxacin,
trimethoprim, ofloxacin, levofloxacin, gatifloxacin, moxifloxacin,
norfloxacin, sodium sulfacetamide, chloramphenicol, tetracycline,
azithromycin, clarithyromycin, trimethoprim sulfate and
bacitracin.
[0065] Yet other examples of therapeutic agents suitable for
inclusion within the disclosed nanoparticles are non-steroidal
(NSAIDs) and steroidal anti-inflammatory agents (generally referred
to as anti inflammatory agents (including both COX-1 and COX-2
inhibitors)). Examples include, but are not limited to,
corticosteroids, medrysone, prednisolone, prednisolone acetate,
prednisolone sodium phosphate, fluormetholone, dexamethasone,
dexamethasone sodium phosphate, betamethasone, fluoromethasone,
antazoline, fluorometholone acetate, rimexolone, loteprednol
etabonate, diclofenac (diclofenac sodium), ketorolac, ketorolac
tromethamine, hydrocortisone, bromfenac, flurbiprofen, antazoline
and xylometazoline.
[0066] Other therapeutic agents that can be incorporated into
nanoparticles as disclosed herein include anti-histamines, mast
cell stabilizers and other anti-allergy agents. Examples include,
but are not limited, cromolyn sodium, lodoxamide tromethamine,
olopatadine HCl, nedocromil sodium, ketotifen fumurate,
levocabastine HCL, azelastine HCL, pemirolast (pemirolast
potassium), epinastine HCL, naphazoline HCL, emedastine,
antazoline, pheniramine, sodium cromoglycate, N-acetyl-aspartyl
glutamic acid and amlexanox.
[0067] Other non-limiting examples of potentially suitable
therapeutic agents for incorporation into nanoparticles include
anti-cancer agents such as 5-fluorouracil (5-FU), CPT-11,
10-hydroxy-7-ethylcamptothecin (SN38), S-1 capecitabine, ftorafur,
5'deoxyfluorouridine, UFT, eniluracil, deoxycytidine,
5-azacytosine, 5-azadeoxycytosine, allopurinol, 2-chloroadenosine,
aminopterin, methylene-10-deazaminopterin (MDAM), oxaplatin,
picoplatin, tetraplatin, satraplatin, platinum-DACH, ormaplatin,
CI-973, JM-216, and analogs thereof, 9-aminocamptothecin,
10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin,
TAS 103, L-phenylalanine mustard, ifosphamidemefosphamide,
trophosphamide carmustine, epothilones A-E, tomudex,
6-mercaptopurine, 6-thioguanine, karenitecin, acyclovir,
valacyclovir, ganciclovir, amantadine, rimantadine, lamivudine,
zidovudine, bevacizumab, trastuzumab, rituximab,20-epi-1.alpha., 25
dihydroxyvitamin D3, 4-ipomeanol, 5-ethynyluracil, 9-dihydrotaxol,
abiraterone, acivicin, aclarubicin, acodazole hydrochloride,
acronine, acylfiilvene, adecypenol, adozelesin, aldesleukin, all-tk
antagonists, altretamine, ambamustine, ambomycin, ametantrone
acetate, amidox, amifostine, aminoglutethimide, aminolevulinic
acid, amrubicin, amsacrine, anagrelide, anastrozole,
andrographolide, angiogenesis inhibitors, antagonist D, antagonist
G, antarelix, anthramycin, anti-dorsalizing morphogenetic
protein-1, antiestrogen, antineoplaston, antisense
oligonucleotides, aphidicolin glycinate, apoptosis gene modulators,
apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine
deaminase, asparaginase, asperlin, asulacrine, atamestane,
atrimustine, axinastatin 1, axinastatin 2, axinastatin 3,
azacitidine, azasetron, azatoxin, azatyrosine, azetepa, azotomycin,
baccatin III derivatives, balanol, batimastat, benzochlorins,
benzodepa, benzoylstaurosporine, beta lactam derivatives,
beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor,
bicalutamide, bisantrene, bisantrene hydrochloride,
bisazuidinylspermine, bisnafide, bisnafide dimesylate, bistratene
A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists,
breflate, brequinar sodium, bropirimine, budotitane, busulfan,
buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C,
calusterone, camptothecin derivatives, canarypox IL-2,
capecitabine, caraceraide, carbetimer, carboplatin,
carboxamide-amino-triazole, carboxyamidotriazole, carest M3,
carmustine, earn 700, cartilage derived inhibitor, carubicin
hydrochloride, carzelesin, casein kinase inhibitors,
castanosperrnine, cecropin B, cedefingol, cetrorelix, chlorambucil,
chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin,
cisplatin, cis-porphyrin, cladribine, clomifene analogs,
clotrimazole, collismycin A, collismycin B, combretastatin A4,
combretastatin analog, conagenin, crambescidin 816, crisnatol,
crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives,
curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam,
cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin
hydrochloride, decitabine, dehydrodidemnin B, deslorelin,
dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil,
dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox,
diethyhiorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl
spiromustine, docetaxel, docosanol, dolasetron, doxifluridine,
doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene
citrate, dromostanolone propionate, dronabinol, duazomycin,
duocannycin SA, ebselen, ecomustine, edatrexate, edelfosine,
edrecolomab, eflomithine, eflomithine hydrochloride, elemene,
elsarnitrucin, emitefur, enloplatin, enpromate, epipropidine,
epirubicin, epirubicin hydrochloride, epristeride, erbulozole,
erythrocyte gene therapy vector system, esorubicin hydrochloride,
estramustine, estramustine analog, estramustine phosphate sodium,
estrogen agonists, estrogen antagonists, etanidazole, etoposide,
etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole
hydrochloride, fazarabine, fenretinide, filgrastim, finasteride,
flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,
fludarabine phosphate, fluorodaunorunicin hydrochloride,
fluorouracil, fluorocitabine, forfenimex, formestane, fosquidone,
fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin,
gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors,
gemcitabine, gemcitabine hydrochloride, glutathione inhibitors,
hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea,
hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride,
idoxifene, idramantone, ifosfamide, ihnofosine, ilomastat,
imidazoacridones, imiquimod, immunostimulant peptides, insulin-like
growth factor-1 receptor inhibitor, interferon agonists, interferon
alpha-2A, interferon alpha-2B, interferon alpha-N1, interferon
alpha-N3, interferon beta-IA, interferon gamma-IB, interferons,
interleukins, iobenguane, iododoxorubicin, iproplatm, irinotecan,
irinotecan hydrochloride, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-N triacetate, lanreotide, lanreotide acetate,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,
leukemia inhibiting factor, leukocyte alpha interferon, leuprolide
acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole,
liarozole, liarozole hydrochloride, linear polyamine analog,
lipophilic disaccharide peptide, lipophilic platinum compounds,
lissoclinamide, lobaplatin, lombricine, lometrexol, lometrexol
sodium, lomustine, lonidamine, losoxantrone, losoxantrone
hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium
texaphyrin lysofylline, lytic peptides, maitansine, mannostatin A,
marimastat, masoprocol, maspin, matrilysin inhibitors, matrix
metalloproteinase inhibitors, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, merbarone, mercaptopurine, meterelin, methioninase,
methotrexate, methotrexate sodium, metoclopramide, metoprine,
meturedepa, microalgal protein kinase C uihibitors, MIF inhibitor,
mifepristone, miltefosine, mirimostim, mismatched double stranded
RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone,
mitolactol, mitomycin, mitomycin, mitomycin analogs, initonafide,
mitosper, mitotane, mitotoxin fibroblast growth factor-saporin,
mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid a/myobacterium cell wall SK, mopidamol, multiple drug
resistance gene inhibitor, multiple tumor suppressor 1-based
therapy, mustard anticancer agent, mycaperoxide B, mycobacterial
cell wall extract, mycophenolic acid, myriaporone,
n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine,
napavin, naphterpin, nartograstim, nedaplatin, nemorubicin,
neridronic acid, neutral endopeptidase, nilutamide, nisamycin,
nitric oxide modulators, nitroxide antioxidant, nitrullyn,
nocodazole, nogalamycin, n-substituted benzamides,
06-benzylguanine, octreotide, okicenone, oligonucleotides,
onapristone, ondansetron, oracin, oral cytokine inducer,
ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran,
paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine,
palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,
parabactin, pazelliptine, pegaspargase, peldesine, peliomycin,
pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole,
peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin,
piritrexim, piroxantrone hydrochloride, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, plicamycin, plomestane,
porfimer sodium, porfiromycin, prednimustine, procarbazine
hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic
carcinoma antiandrogen, proteasome inhibitors, protein A-based
immune modulator, protein kinase C inhibitor, protein tyrosine
phosphatase inhibitors, purine nucleoside phosphorylase inhibitors,
puromycin, puromycin hydrochloride, purpurins, pyrazorurin,
pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene
conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl
protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor,
retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin,
riboprine, ribozymes, RH retinamide, rogletimide, rohitukine,
romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, safingol
hydrochloride, saintopin, sarcnu, sarcophytol A, sargramostim, SDI1
mimetics, semustine, senescence derived inhibitor 1, sense
oligonucleotides, signal transduction inhibitors, signal
transduction modulators, simtrazene, single chain antigen binding
protein, sizofuran, sobuzoxane, sodium borocaptate, sodium
phenylacetate, solverol, somatomedin binding protein, sonermin,
sparfosafe sodium, sparfosic acid, sparsomycin, spicamycin D,
spirogermanium hydrochloride, spiromustine, spiroplatin,
splenopentin, spongistatin 1, squalamine, stem cell inhibitor,
stem-cell division inhibitors, stipiamide, streptonigrin,
streptozocin, stromelysin inhibitors, sulfinosine, sulofenur,
superactive vasoactive intestinal peptide antagonist, suradista,
suramin, swainsonine, synthetic glycosaminoglycans, talisomycin,
tallimustine, tamoxifen methiodide, tauromustine, tazarotene,
tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors,
teloxantrone hydrochloride, temoporfin, temozolomide, teniposide,
teroxirone, testolactone, tetrachlorodecaoxide, tetrazomine,
thaliblastine, thalidomide, thiamiprine, thiocoraline, thioguanine,
thiotepa, thrombopoietin, thrombopoietin mimetic, thymalfasin,
thymopoietin receptor agonist, thymotrinan, thyroid stimulating
hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine,
titanocene dichloride, topotecan hydrochloride, topsentin,
toremifene, toremifene citrate, totipotent stem cell factor,
translation inhibitors, trestolone acetate, tretinoin,
triacetyluridine, triciribine, triciribine phosphate, trimetrexate,
trimetrexate glucuronate, triptorelin, tropisetron, tubulozole
hydrochloride, turosteride, tyrosine kinase inhibitors,
tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, velaresol, veramine,
verdins, verteporfin, vinblastine sulfate, vincristine sulfate,
vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate
sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate,
vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin,
vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin
stimalamer, or zorubicin hydrochloride and combinations
thereof.
[0068] In one embodiment, the nanoparticles of this invention can
contain siRNA as a therapeutic agent. Preferably, the siRNA
molecule has a length from about 10-50 or more nucleotides. More
preferably, the siRNA molecule has a length from about 15-45
nucleotides. Even more preferably, the siRNA molecule has a length
from about 19-40 nucleotides. Even more preferably, the siRNA
molecule has a length of from about 21-23 nucleotides. The siRNA of
the invention preferably mediates RNAi against a target mRNA. The
siRNA molecule can be designed such that every residue is
complementary to a residue in the target molecule. Alternatively,
one or more substitutions can be made within the molecule to
increase stability and/or enhance processing activity of said
molecule. Substitutions can be made within the strand or can be
made to residues at the ends of the strand.
[0069] siRNA molecules used as therapeutic agents in the disclosed
nanoparticles can be modified to improve stability either in vivo
or in vitro. In order to enhance the stability, the 3'-residues may
be stabilized against degradation, e.g., they may be selected such
that they consist of purine nucleotides, particularly adenosine or
guanosine nucleotides. Alternatively, substitution of pyrimidine
nucleotides by modified analogues, e.g., substitution of uridine by
2'-deoxythymidine is tolerated and does not affect the efficiency
of RNA interference. For example, the absence of a 2'hydroxyl may
significantly enhance the nuclease resistance of the siRNAs.
[0070] Commercially available design tools and kits, such as those
available from Ambion, Inc. (Austin, Tex.), and the Whitehead
Institute of Biomedical Research at MIT (Cambridge, Mass.) allow
for the design and production of siRNA. By way of example, a
desired mRNA sequence can be entered into a sequence program that
will generate sense and antisense target strand sequences. These
sequences can then be entered into a program that determines the
sense and antisense siRNA oligonucleotide templates. The programs
can also be used to add, e.g., hairpin inserts or T1 promoter
primer sequences. Kits also can then be employed to build siRNA
expression cassettes.
[0071] In various embodiments, siRNAs are synthesized in vivo, in
situ, and in vitro. Endogenous RNA polymerase of the cell may
mediate transcription in vivo or in situ, or cloned RNA polymerase
can be used for transcription in vivo or in vitro. For
transcription from a transgene in vivo or an expression construct,
a regulatory region (e.g., promoter, enhancer, silencer, splice
donor and acceptor, polyadenylation) may be used to transcribe the
siRNAs. Inhibition may be targeted by specific transcription in an
organ, tissue, or cell type; stimulation of an environmental
condition (e.g., infection, stress, temperature, chemical
inducers); and/or engineering transcription at a developmental
stage or age. A transgenic organism that expresses siRNAs from a
recombinant construct may be produced by introducing the construct
into a zygote, an embryonic stem cell, or another multipotent cell
derived from the appropriate organism.
[0072] In one embodiment, the siRNA molecules target mRNA encoding
at least one a cellular protein (e.g., a nuclear, cytoplasmic,
transmembrane, or membrane-associated protein). In another
embodiment, the siRNA molecules target mRNA encoding one or more
extracellular protein (e.g., an extracellular matrix protein or
secreted protein). Thus, target mRNA can encode developmental
proteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt
family members, Pax family members, Winged helix family members,
Hox family members, cytokines/lymphokines and their receptors,
growth/differentiation factors and their receptors,
neurotransmitters and their receptors); oncogene-encoded proteins
(e.g., ABLI, BCLI, BCL2, BCL6, CBFA2. CBL, CSFIR, ERBA, ERBB,
EBRB2, ERBB2, ERBB3, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS,
JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1,
PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins
(e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, or
WTI); and enzymes (e.g., ACC synthases and oxidases, ACP
desaturases and hydroxylases, ADPglucose pyrophorylases, acetylases
and deacetylases, ATPases, alcohol dehydrogenases, amylases,
amyloglucosidases, catalases, cellulases, chalcone synthases,
chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and
RNA polymerases, galactosidases, glucanases, glucose oxidases,
granule-bound starch synthases, GTPases, helicases, hemicellulases,
integrases, inulinases, invertases, isomerases, kinases, lactases,
lipases, lipoxygenases, lysozymes, nopaline synthases, octopine
synthases, pectinesterases, peroxidases, phosphatases,
phospholipases, phosphorylases, phytases, plant growth regulator
synthases, polygalacturonases, proteinases and peptidases,
pullanases, recombinases, reverse transcriptases, RUBISCOs,
topoisomerases or xylanases). Target mRNA can also encode proteins
involved in tumor growth (including vascularization) or in
metastatic activity or potential (e.g., cell surface receptors and
their ligands). Target mRNA can also encode one or more secreted
protein, cell cycle regulatory protein, gene regulatory protein,
apoptosis regulatory protein, or proteins involved in the immune
response, inflammation, complement cascade, or clotting systems.
Additional examples of target mRNA against which siRNA constructs
can designed include c-myc, c-myb, mdm2, PKA-I (protein kinase A
type I), Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclin
dependent kinases (cdks), telomerase, PDGF/sis and mos. siRNA can
also be used to target the mRNA encoded by a fusion gene that
results from chromosomal translocation, for example, the Bcr/Abl
fusion oncogene. siRNA can also be targeted against mRNA encoding
proteins such as cyclin dependent kinases, proliferating cell
nuclear antigen (PCNA), transforming growth factor-beta (TGF-beta),
nuclear factor kappa B (NF-.kappa.B), E2F, HER-2/neu, PKA,
TGF-alpha, EGFR, TGF-beta, IGFIR, P12, MDM2, VEGF, MDR,
transferring, ferritin, ferritin receptor, transferrin receptor,
IRE, C-fos, HSP27, metallothionein.
Targeting Agents
[0073] As noted above, nanoparticles produced by the instant
invention can also incorporate one or more targeting agent via the
functionalized PEG moieties. Suitable targeting agents include, for
example, antibodies and polypeptides that bind to polypeptides that
are commonly overexpressed by tumor or cancer cells. Non-limiting
examples of such polypeptides are epidermal growth factor receptor
(EGFR), somatostatin receptor (SSTR), insulin-like growth factor
receptor, folic acid-receptor, HER.sup.2 receptor, interleukin-13
receptor, gastrin-releasing peptide receptor, CD30, vasoactive
intestinal peptide receptor, gastrin receptor, prostate-specific
antigen, and the estrogen receptor.
[0074] Another example of a targeting agents suitable for coupling
to the disclosed nanoparticles via functionalized PEG are small
molecule ligands. Such small molecule ligands can be used to target
cancers that express particular target proteins. For example, for
tumors or cancers that express prostate specific membrane antigen
(PSMA) (including, but not limited to, prostate cancer, non-small
cell lung cancer, colorectal carcinoma, and glioblastoma, and solid
tumors expressing PSMA in the tumor neovasculature), PSMA ligands
can be used. In some embodiments, the low-molecular weight PSMA
ligand is of the Formulae I, II, III or IV:
##STR00014##
[0075] and enantiomers, stereoisomers, rotamers, tautomers,
diastereomers, or racemates thereof;
[0076] wherein
[0077] m and n are each, independently, 0, 1, 2 or 3;
[0078] p is 0 or 1;
[0079] R.sup.1, R.sup.2, R.sup.4 and R.sup.5 are each,
independently, selected from the group consisting of substituted or
unsubstituted alkyl (e.g., C.sub.1-10-alkyl, C.sub.1-6-alkyl, or
C.sub.i-4-alkyl), substituted or unsubstituted aryl (e.g., phenyl
or pyrdinyl), and any combination thereof; and
[0080] R.sup.3 is H or C.sub.1-6-alkyl (e.g., CH.sub.3).
[0081] For compounds of Formulae I, II, III and IV, R.sup.1,
R.sup.2, R.sup.4 and R.sup.5 comprise points of attachment to the
nanoparticle within the PEG portion of the nanoparticle. The point
of attachment may be formed by a covalent bond, ionic bond,
hydrogen bond, a bond formed by adsorption including chemical
adsorption and physical adsorption, a bond formed from van der
Waals bonds, or dispersion forces. For example, if R.sup.1,
R.sup.2, R.sup.4 or R.sup.5 are defined as an aniline or
C.sub.1-6-alkyl-NH.sub.2 group, any hydrogen (e.g., an amino
hydrogen) of these functional groups could be removed such that the
low-molecular weight PSMA ligand is covalently bound to the
polymeric matrix (e.g., the PEG-block of the polymeric matrix) of
the nanoparticle. As used herein, the term "covalent bond" refers
to a bond between two atoms formed by sharing at least one pair of
electrons.
[0082] In particular embodiments of the Formulae I, II, III or IV,
R.sup.1, R.sup.2, R.sup.4 and R.sup.5 are each, independently,
C.sub.1-6-alkyl or phenyl, or any combination of C.sub.1-6-alkyl or
phenyl, which are independently substituted one or more times with
OH, SH, NH.sub.2, or CO.sub.2H, and wherein the alkyl group may be
interrupted by N(H), S or O. In another embodiment, R.sup.1,
R.sup.2, R.sup.4 and R.sup.5 are each, independently, CH.sub.2-Ph,
(CH.sub.2).sub.2--SH, CH.sub.2--SH,
(CH.sub.2).sub.2C(H)(NH.sub.2)CO.sub.2H,
CH.sub.2C(H)(NH.sub.2)CO.sub.2H, CH(NH.sub.2)CH.sub.2CO.sub.2H,
(CH.sub.2).sub.2C(H)(SH)CO.sub.2H, CH.sub.2--N(H)-Ph,
O--CH.sub.2-Ph, or O--(CH.sub.2).sub.2-Ph, wherein each Ph may be
independently substituted one or more times with OH, NH.sub.2,
CO.sub.2H or SH. For these formulae, the NH.sub.2, OH or SH groups
serve as the point of covalent attachment to the nanoparticle
(e.g., --N(H)-PEG, --O-PEG, or S-PEG).
[0083] In still another embodiment, the low-molecular weight PSMA
ligand is selected from the group consisting of
##STR00015##
[0084] and enantiomers, stereoisomers, rotamers, tautomers,
diastereomers, or racemates thereof, and wherein the NH.sub.2, OH
or SH groups serve as the point of covalent attachment to the
nanoparticle (e.g., --N(H)-PEG, --O-PEG, or S-PEG).
[0085] In another embodiment, the low-molecular weight PSMA ligand
is selected from the group consisting of
##STR00016##
and enantiomers, stereoisomers, rotamers, tautomers, diastereomers,
or racemates thereof, wherein R is independently selected from the
group consisting of NH.sub.2, SH, OH, CO.sub.2H, C.sub.1-6-alkyl
that is substituted with NH.sub.2, SH, OH or CO.sub.2H, and phenyl
that is substituted with NH.sub.2, SH, OH or CO.sub.2H, and wherein
R serves as the point of covalent attachment to the nanoparticle
(e.g., --N(H)-PEG, S-PEG, --O-PEG, or CO.sub.2-PEG).
[0086] In another embodiment, the low-molecular weight PSMA ligand
is selected from the group consisting of
##STR00017##
and enantiomers, stereoisomers, rotamers, tautomers, diastereomers,
or racemates thereof, wherein the NH.sub.2 or CO.sub.2H groups
serve as the point of covalent attachment to the nanoparticle
(e.g., --N(H)-PEG, or CO.sub.2-PEG). These compounds may be further
substituted with NH.sub.2, SH, OH, CO.sub.2H, C.sub.1-6-alkyl that
is substituted with NH.sub.2, SH, OH or CO.sub.2H, or phenyl that
is substituted with NH.sub.2, SH, OH or CO.sub.2H, wherein these
functional groups can also serve as the point of covalent
attachment to the nanoparticle.
[0087] In another embodiment, the low-molecular weight PSMA ligand
is
##STR00018##
[0088] and enantiomers, stereoisomers, rotamers, tautomers,
diastereomers, or racemates thereof, wherein n is 1, 2, 3, 4, 5 or
6. For this ligand, the NH.sub.2 group serves as the point of
covalent attachment to the nanoparticle (e.g., --N(H)-PEG).
[0089] In still another embodiment, the low-molecular weight PSMA
ligand is
##STR00019##
and enantiomers, stereoisomers, rotamers, tautomers, diastereomers,
or racemates thereof. Particularly, the butyl-amine compound has
the advantage of ease of synthesis, especially because of its lack
of a benzene ring. Furthermore, without wishing to be bound by
theory, the butyl-amine compound will likely break down into
naturally occurring molecules (i.e., lysine and glutamic acid),
thereby minimizing toxicity concerns.
[0090] For these ligands, the NH.sub.2 groups serve as the point of
covalent attachment to the nanoparticle (e.g., --N(H)-PEG).
Accordingly, the present invention provides the low-molecular
weight PSMA ligands shown above, wherein the amine substituents of
the compounds are covalently bound to polyethylene glycol), e.g.,
the
##STR00020##
compounds: wherein n is 20 to 1720.
[0091] Another aspect of the invention provides for a PEG polymer
conjugated to a targeting agents and/or bioactive
moieties/therapeutic agents as disclosed herein.
Nanoparticle Compositions and Uses Thereof
[0092] The nanoparticles produced in accordance with the disclosed
methods can be administered alone or in a composition. When
nanoparticles are administered as a composition, the composition
can be a pharmaceutical (e.g., physiologically acceptable)
composition. The composition comprises a carrier (e.g., a
pharmaceutically or physiologically acceptable carrier) and the
nanoparticles. Any suitable carrier (e.g., water, saline, and PBS)
can be used within the context of the invention, and such carriers
are well known in the art. The choice of carrier will be
determined, in part, by the particular site to which the
composition is to be administered and the particular method used to
administer the composition. Suitable carriers, as well as other
components suitable for use in the composition of the invention,
are known in the art (e.g., Remington's Pharmaceutical Sciences,
17th ed., (Mack Publishing Company, Philadelphia, Pa.: 1985)).
Additionally, the composition can comprise additional active
agents, such as anti-cancer/chemotherapeutic agents.
[0093] The nanoparticles and composition thereof can be
administered to a subject to treat or prevent particular disorders
and diseases. Non-limiting examples of diseases or disorders that
can be treated in accordance with the subject invention include
cancer, such as lung cancer, breast cancer, prostate cancer, head
and neck cancer, ovarian cancer, skin cancer, testicular cancer,
pancreatic cancer, esophageal cancer, colorectal cancer, kidney
cancer, cervical cancer, gastrointestinal cancer, and combinations
thereof. The nanoparticles or the composition thereof preferably
are administered to the subject in a therapeutically effective
amount.
[0094] A therapeutically effective amount refers to an amount of
the nanoparticles necessary to treat or prevent the particular
disease or disorder. For example nanoparticles disclosed herein can
be used to inhibit tumor growth, inhibit or reduce proliferation,
invasiveness, or metastasis of tumor or cancer cells, slow the
growth of tumors or cancers or reduce the size of tumors.
[0095] Any route of administration can be used to deliver the
nanoparticles to the subject. Suitable administration routes
include intramuscular injection, transdermal administration,
inhalation, topical application to tissue (e.g., tumor/cancer
tissue), intratumoral administration, and parenteral administration
(e.g., intravenous, peritoneal, intraarterial, subcutaneous,
rectal, or vaginal, administration). An appropriate administration
route easily can be determined by those skilled in the art.
[0096] As discussed above, compositions comprising nanoparticles
can be useful in the treatment or prevention or amelioration of one
or more symptoms of cancer, particularly cancers that express
prostate specific membrane antigen (PSMA). These cancers include,
but are not limited to, prostate cancer, non-small cell lung
cancer, colorectal carcinoma, and glioblastoma, and solid tumors
expressing PSMA in the tumor neovasculature.
[0097] Various non-limiting embodiments include:
[0098] 1. A method of preparing a nanoparticle comprising: [0099]
providing a targeting agent; [0100] providing a functionalized
poly(ethylene glycol) (PEG) polymer; [0101] providing a targeting
agent ligand; [0102] reacting the functionalized poly(ethylene
glycol) polymer with the targeting agent to form a targeting
agent-PEG polymer complex; and [0103] mixing the targeting
agent-PEG polymer complex with a second polymer and a therapeutic
agent to form a nanoparticle, wherein:
[0104] the PEG polymer has a molecular weight of between
1,000-20,000 Da (e.g., 5,000-20,000, e.g., 10,000-20,000) and, in
some specific embodiments, 5000 Da; and the second polymer has a
molecular weight of 5,000-100,000 Da (e.g., 20,000-70,000, e.g.,
20,000-50,000), or in some specific embodiments, 15,000-30,000
Da;
[0105] 2. The method of embodiment 1, wherein the poly(ethylene
glycol) is hetero-bifunctional and said targeting agent is
covalently bound to the .alpha. terminus of said poly(ethylene
glycol) and at least one polymerization initiating functional group
is present on the .omega. terminus of said poly(ethylene
glycol);
[0106] 3. The method of embodiment 2, wherein said at least one
polymerization initiating functional group is a hydroxyl (--OH)
group or an amine (--NH.sub.2) group at the free .omega.
terminus;
[0107] 4. The method of embodiment 1, 2 or 3, wherein the second
polymer comprises a blend of two or more polymers and contains at
least one functional group that reacts the functional group present
at the free .omega. terminus of said poly(ethylene glycol) and said
at least one functional group of said blend of two or more polymers
is a hydroxyl group, a NHS group or an amine group;
[0108] 5. The method of embodiment 1, wherein the second polymer or
the copolymer is a polyester copolymer that contains at least one
functional group selected from a hydroxyl group, a NHS group or an
amine group and that reacts the functional group present at the
free .omega. terminus of said poly(ethylene glycol);
[0109] 6. The method of embodiment 4, wherein the second polymer or
the copolymer is a polyester copolymer that contains at least one
functional group selected from a hydroxyl group, a NHS group or an
amine group and that reacts the functional group present at the
free .omega. terminus of said poly(ethylene glycol);
[0110] 7. The method of embodiment 6, wherein said polyester
copolymer comprises a heteropolymer or a homopolymer;
[0111] 8. The method of embodiment 6, wherein said heteropolymer
comprises lactic acid and glycolic acid units or poly(lactic
acid-co-glycolic acid) and poly(lactide-co-glycolide) units (PLGA);
and said homopolymer comprises glycolic acid units (PGA), lactic
acid units (PLA), poly-L-lactic acid units, poly-D-lactic acid
units, poly-D,L-lactic acid units, poly-L-lactide units,
poly-D-lactide units or poly-D,L-lactide units;
[0112] 9. The method of embodiment 6, wherein said polyester
copolymer is selected from polyhydroxyacids; PEGylated polymers and
copolymers of lactide units and glycolide units, PEGylated PLA,
PEGylated PGA, PEGylated PLGA, polyanhydrides, poly(ortho ester)
PEGylated poly(ortho ester), poly(caprolactone), PEGylated
poly(caprolactone), polylysine, PEGylated polylysine, poly(ethylene
inline), PEGylated poly(ethylene imine),
poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester), poly[a-(4-aminobutyl)-L-glycolic
acid] or derivatives thereof;
[0113] 10. The method of embodiment 1, wherein free carboxylic acid
groups or free hydroxyl groups on said targeting agent are
protected prior to reacting the functionalized poly(ethylene
glycol) polymer with the targeting agent to form a targeting
agent-PEG polymer complex;
[0114] 11. The method of embodiment 4, wherein said functional
group of said second polymer is an amine group that reacts with a
hydroxyl group or carboxylic acid group on said targeting agent-PEG
complex;
[0115] 12. The method of embodiment 4, wherein said functional
group of said second polymer is a hydroxyl group or an NHS group
that reacts with an amine group on said targeting agent-PEG
complex;
[0116] 13. The method of embodiment 6, wherein said functional
group of said second polymer or said copolymer is an amine group
that reacts with a hydroxyl group or carboxylic acid group on said
targeting agent-PEG complex;
[0117] 14. The method of embodiment 6, wherein said functional
group of said second polymer or said copolymer is a NHS or hydroxyl
group that reacts with an amine group on said targeting agent-PEG
complex;
[0118] 15. The method of embodiments 1-14, wherein said second
polymer is a blend of at least two polymers which can be the same
or different polymer, wherein the first of said at least two
polymers contains at least one hydroxyl group or an NHS group as a
functional group and the second of said at least two polymers
contains at least one amine group as said functional group;
[0119] 16. The method of embodiment 1-15, wherein said therapeutic
agent is an antibiotic, anti-cancer agent, antiviral agent,
anti-inflammatory agent a diagnostic agent, a vaccine antigen or a
nutraceutical;
[0120] 17. The method of embodiment 16, wherein said therapeutic
agent is/are penicillins, aminopenicillins, penicillins in
conjunction with penicillinase inhibitor and/or anti-fungal agents,
cephalosporins, cephamycins, carbapenems, fluoroquinolones,
tetracyclines, macrolides, aminoglycosides, erythromycin,
bacitracin zinc, polymyxin, polymyxin B sulfates, neomycin,
gentamycin, tobramycin, gramicidin, ciprofloxacin, trimethoprim,
ofloxacin, levofloxacin, gatifloxacin, moxifloxacin, norfloxacin,
sodium sulfacetamide, chloramphenicol, tetracycline, azithromycin,
clarithyromycin, trimethoprim sulfate, bacitracin, corticosteroids,
medrysone, prednisolone, prednisolone acetate, prednisolone sodium
phosphate, fluormetholone, dexamethasone, dexamethasone sodium
phosphate, betamethasone, fluoromethasone, antazoline,
fluorometholone acetate, rimexolone, loteprednol etabonate,
diclofenac (diclofenac sodium), ketorolac, ketorolac tromethamine,
hydrocortisone, bromfenac, flurbiprofen, antazoline,
xylometazoline, cromolyn sodium, lodoxamide tromethamine,
olopatadine HCl, nedocromil sodium, ketotifen fumurate,
levocabastine HCL, azelastine HCL, pemirolast (pemirolast
potassium), epinastine HCL, naphazoline HCL, emedastine,
antazoline, pheniramine, sodium cromoglycate, N-acetyl-aspartyl
glutamic acid, amlexanox, 5-fluorouracil (5-FU), CPT-11,
10-hydroxy-7-ethylcamptothecin (SN38), S-I capecitabine, ftorafur,
5' deoxyfluorouridine, UFT, eniluracil, deoxycytidine,
5-azacytosine, 5-azadeoxycytosine, allopurinol, 2-chloroadenosine,
aminopterin, methylene-10-deazaminopterin (MDAM), oxaplatin,
picoplatin, tetraplatin, satraplatin, platinum-DACH, ormaplatin,
CI-973, JM-216, and analogs thereof, 9-aminocamptothecin,
10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin,
TAS 103, L-phenylalanine mustard, ifosphamidemefosphamide,
trophosphamide carmustine, epothilones A-E, tomudex,
6-mercaptopurine, 6-thioguanine, karenitecin, acyclovir,
valacyclovir, ganciclovir, amantadine, rimantadine, lamivudine,
zidovudine, bevacizumab, trastuzumab, rituximab,20-epi-1.alpha., 25
dihydroxyvitamin D3, 4-ipomeanol, 5-ethynyluracil, 9-dihydrotaxol,
abiraterone, acivicin, aclarubicin, acodazole hydrochloride,
acronine, acylfiilvene, adecypenol, adozelesin, aldesleukin, all-tk
antagonists, altretamine, ambamustine, ambomycin, ametantrone
acetate, amidox, amifostine, aminoglutethimide, aminolevulinic
acid, amrubicin, amsacrine, anagrelide, anastrozole,
andrographolide, angiogenesis inhibitors, antagonist D, antagonist
G, antarelix, anthramycin, anti-dorsalizing morphogenetic
protein-1, antiestrogen, antineoplaston, antisense
oligonucleotides, aphidicolin glycinate, apoptosis gene modulators,
apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine
deaminase, asparaginase, asperlin, asulacrine, atamestane,
atrimustine, axinastatin 1, axinastatin 2, axinastatin 3,
azacitidine, azasetron, azatoxin, azatyrosine, azetepa, azotomycin,
baccatin III derivatives, balanol, batimastat, benzochlorins,
benzodepa, benzoylstaurosporine, beta lactam derivatives,
beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor,
bicalutamide, bisantrene, bisantrene hydrochloride,
bisazuidinylspermine, bisnafide, bisnafide dimesylate, bistratene
A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists,
breflate, brequinar sodium, bropirimine, budotitane, busulfan,
buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C,
calusterone, camptothecin derivatives, canarypox IL-2,
capecitabine, caraceraide, carbetimer, carboplatin,
carboxamide-amino-triazole, carboxyamidotriazole, carest M3,
carmustine, earn 700, cartilage derived inhibitor, carubicin
hydrochloride, carzelesin, casein kinase inhibitors,
castanosperrnine, cecropin B, cedefingol, cetrorelix, chlorambucil,
chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin,
cisplatin, cis-porphyrin, cladribine, clomifene analogs,
clotrimazole, collismycin A, collismycin B, combretastatin A4,
combretastatin analog, conagenin, crambescidin 816, crisnatol,
crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives,
curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam,
cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin
hydrochloride, decitabine, dehydrodidemnin B, deslorelin,
dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil,
dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox,
diethyhiorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl
spiromustine, docetaxel, docosanol, dolasetron, doxifluridine,
doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene
citrate, dromostanolone propionate, dronabinol, duazomycin,
duocannycin SA, ebselen, ecomustine, edatrexate, edelfosine,
edrecolomab, eflomithine, eflomithine hydrochloride, elemene,
elsarnitrucin, emitefur, enloplatin, enpromate, epipropidine,
epirubicin, epirubicin hydrochloride, epristeride, erbulozole,
erythrocyte gene therapy vector system, esorubicin hydrochloride,
estramustine, estramustine analog, estramustine phosphate sodium,
estrogen agonists, estrogen antagonists, etanidazole, etoposide,
etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole
hydrochloride, fazarabine, fenretinide, filgrastim, finasteride,
flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,
fludarabine phosphate, fluorodaunorunicin hydrochloride,
fluorouracil, fluorocitabine, forfenimex, formestane, fosquidone,
fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin,
gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors,
gemcitabine, gemcitabine hydrochloride, glutathione inhibitors,
hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea,
hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride,
idoxifene, idramantone, ifosfamide, ihnofosine, ilomastat,
imidazoacridones, imiquimod, immunostimulant peptides, insulin-like
growth factor-1 receptor inhibitor, interferon agonists, interferon
alpha-2A, interferon alpha-2B, interferon alpha-N1, interferon
alpha-N3, interferon beta-IA, interferon gamma-IB, interferons,
interleukins, iobenguane, iododoxorubicin, iproplatm, irinotecan,
irinotecan hydrochloride, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-N triacetate, lanreotide, lanreotide acetate,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,
leukemia inhibiting factor, leukocyte alpha interferon, leuprolide
acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole,
liarozole, liarozole hydrochloride, linear polyamine analog,
lipophilic disaccharide peptide, lipophilic platinum compounds,
lissoclinamide, lobaplatin, lombricine, lometrexol, lometrexol
sodium, lomustine, lonidamine, losoxantrone, losoxantrone
hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium
texaphyrin lysofylline, lytic peptides, maitansine, mannostatin A,
marimastat, masoprocol, maspin, matrilysin inhibitors, matrix
metalloproteinase inhibitors, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, merbarone, mercaptopurine, meterelin, methioninase,
methotrexate, methotrexate sodium, metoclopramide, metoprine,
meturedepa, microalgal protein kinase C uihibitors, MIF inhibitor,
mifepristone, miltefosine, mirimostim, mismatched double stranded
RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone,
mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide,
mitosper, mitotane, mitotoxin fibroblast growth factor-saporin,
mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid a/myobacterium cell wall SK, mopidamol, multiple drug
resistance gene inhibitor, mustard anticancer agent, mycaperoxide
B, mycobacterial cell wall extract, mycophenolic acid, myriaporone,
n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine,
napavin, naphterpin, nartograstim, nedaplatin, nemorubicin,
neridronic acid, neutral endopeptidase, nilutamide, nisamycin,
nitric oxide modulators, nitroxide antioxidant, nitrullyn,
nocodazole, nogalamycin, n-substituted benzamides,
06-benzylguanine, octreotide, okicenone, oligonucleotides,
onapristone, ondansetron, oracin, oral cytokine inducer,
ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran,
paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine,
palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,
parabactin, pazelliptine, pegaspargase, peldesine, peliomycin,
pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole,
peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin,
piritrexim, piroxantrone hydrochloride, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, plicamycin, plomestane,
porfimer sodium, porfiromycin, prednimustine, procarbazine
hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic
carcinoma antiandrogen, proteasome inhibitors, protein A-based
immune modulator, protein kinase C inhibitor, protein tyrosine
phosphatase inhibitors, purine nucleoside phosphorylase inhibitors,
puromycin, puromycin hydrochloride, purpurins, pyrazorurin,
pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene
conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl
protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor,
retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin,
riboprine, ribozymes, RH retinamide, RNAi, rogletimide, rohitukine,
romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, safingol
hydrochloride, saintopin, sarcnu, sarcophytol A, sargramostim, SDI1
mimetics, semustine, senescence derived inhibitor 1, sense
oligonucleotides, signal transduction inhibitors, signal
transduction modulators, simtrazene, single chain antigen binding
protein, sizofuran, sobuzoxane, sodium borocaptate, sodium
phenylacetate, solverol, somatomedin binding protein, sonermin,
sparfosafe sodium, sparfosic acid, sparsomycin, spiramycin D,
spirogermanium hydrochloride, spiromustine, spiroplatin,
splenopentin, spongistatin 1, squalamine, stem cell inhibitor,
stem-cell division inhibitors, stipiamide, streptonigrin,
streptozocin, stromelysin inhibitors, sulfinosine, sulofenur,
superactive vasoactive intestinal peptide antagonist, suradista,
suramin, swainsonine, synthetic glycosaminoglycans, talisomycin,
tallimustine, tamoxifen methiodide, tauromustine, tazarotene,
tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors,
teloxantrone hydrochloride, temoporfin, temozolomide, teniposide,
teroxirone, testolactone, tetrachlorodecaoxide, tetrazomine,
thaliblastine, thalidomide, thiamiprine, thiocoraline, thioguanine,
thiotepa, thrombopoietin, thrombopoietin mimetic, thymalfasin,
thymopoietin receptor agonist, thymotrinan, thyroid stimulating
hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine,
titanocene dichloride, topotecan hydrochloride, topsentin,
toremifene, toremifene citrate, totipotent stem cell factor,
translation inhibitors, trestolone acetate, tretinoin,
triacetyluridine, triciribine, triciribine phosphate, trimetrexate,
trimetrexate glucuronate, triptorelin, tropisetron, tubulozole
hydrochloride, turosteride, tyrosine kinase inhibitors,
tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, velaresol, veramine,
verdins, verteporfin, vinblastine sulfate, vincristine sulfate,
vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate
sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate,
vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin,
vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin
stimalamer, or zorubicin hydrochloride, siRNA or any combinations
thereof;
[0121] 18. The method according of embodiment 1-17, wherein said at
least one targeting agent is selected from antibodies, PMSA ligands
and polypeptides that bind to epidermal growth factor receptor
(EGFR), somatostatin receptor (SSTR), insulin-like growth factor
receptor, folic acid-receptor, HER2 receptor, interleukin-13
receptor, gastrin-releasing peptide receptor, CD30, vasoactive
intestinal peptide receptor, gastrin receptor, prostate-specific
antigen, and/or the estrogen receptor;
[0122] 19. The method of embodiment 1-18, wherein said method is
performed in an aqueous medium;
[0123] 20. The method of embodiment 1-18, wherein said method is
performed in an organic solvent;
[0124] 21. A nanoparticle produced according to the methods of
embodiments 1-20;
[0125] 22. A composition comprising the nanoparticle of embodiment
21 and a pharmaceutically acceptable carrier;
[0126] 23. A method of treating a disease comprising the
administration of a nanoparticle according of embodiment 21 to a
subject in an amount sufficient to treat said disease;
[0127] 24. The method of embodiment 23, wherein said disease is
selected from lung cancer, breast cancer, prostate cancer, head and
neck cancer, ovarian cancer, skin cancer, testicular cancer,
pancreatic cancer, esophageal cancer, colorectal cancer, kidney
cancer, cervical cancer, gastrointestinal cancer, viral infections,
bacterial infections or inflammation; and
[0128] 25. A polymer produced according to the method of
embodiments 1-19, said polymer comprising a targeting agent
covalently bound to the .alpha. terminus of a poly(ethylene glycol)
polymer and at least one second polymer covalently bound to said
poly(ethylene glycol) polymer via a functional group present on the
.omega. terminus of said poly(ethylene glycol).
[0129] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated with
the scope of the invention without limitation thereto.
Example 1
Synthesis of HO-PEG-lys-urea-glu (Protected) by Covalent
Conjugation of lys-urea-glu (Protected) to HO-PEG-CO.sub.2H
[0130] Materials used for the synthesis and purification of
HO-PEG-lys-urea-glu (protected) included
.alpha.-hydroxyethyl-.omega.-carboxypentyl, poly(ethylene glycol)
(Average Molecular Weight=5 Kg/mol, Terminal Activity=98%)
(HO-PEG-CO.sub.2H, NOF America Corporation, White Plains, N.Y.);
Diprop-2-en-1-yl
N-{[6-ammonio-1-oxo-1-(prop-2-en-1-yloxy)hexan-2-yl]carbamoyl}glutamate
trifluoroacetate (lys-urea-glu (protected), Organix, Woburn,
Mass.); N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide
(.gtoreq.97.0%) (EDC, Sigma-Aldrich); N-Ethyl Di-isopropyamine
(99.5%) (DIEA, Sigma-Aldrich); N-hydroxysuccinimide (98%) (NHS,
Sigma-Aldrich); Chloroform (Anhydrous, .gtoreq.99%) Sigma-Aldrich);
Ethanol (Absolute, .gtoreq.99.5%) (Sigma-Aldrich).
[0131] Instrumentation used for the in-process characterization and
work-up of HO-PEG-lys-urea-glu (protected) included Bruker 400 MHz
Nuclear Magnetic Resonance Spectrometer for Proton NMR
Spectroscopic Analysis; Vacuum atmospheres dry nitrogen glove box
(<1 ppm water); Edwards RV5 Vacuum Pump and a VWR Oven for
Vacuum Drying; Ultrafiltration Diafiltration (UFDF) System based on
a peristaltic pump (Masterson), Regenerated Cellulose Membrane
filter (Millipore, Surface area=0.1 m.sup.2, MWCO 3 kDa), and
expanded PTFE tubing (Chem-Sure); Rotary Evaporator (Buchi);
Millipore 2 L Vacuum Filtration Assembly and Millipore 0.2 um
Fluoropore (PTFE) Membrane Filters; Waters Size Exclusion
Chromatography system equipped with Refractive Index Detector and
Waters HR1, HR3 and HR4 columns (7.8.times.300 mm, Molecular Weight
Range 100-5000 Da, 500-30,000 Da, 5000-600,000 Da, respectively).
Number and Weight Average Molar Mass was determined using
Poly(ethylene glycol) calibration standards from Shodex Standards,
(Kawasaki, Japan) of Peak Molar Mass (Mp=960, 1400, 4290, 7130,
12900, 20600 Da).
[0132] Experimental procedure: All reagents (HO-PEG-CO.sub.2H,
lys-urea-glu (protected), EDC, NHS, DIEA and anhydrous chloroform)
were transferred to a dry nitrogen glove box and subsequently
weighed or transferred (using volumetric pipettes or microliter
syringes) in 20 mL reaction vials under argon at room temperature.
NHS (230 mg, 2 mmol) was added to a solution of HO-PEG-CO.sub.2H (1
g, 0.2 mmol) in anhydrous chloroform (5 mL) (solution A). GL2P
(1.106 g, 2 mmol) was dissolved in anhydrous chloroform (5 mL),
then EDC (354 uL, 2 mmol) and DIEA (685 uL, 4 mmol) were added to
this solution (solution B). Solution B was added to solution A and
the reaction vial (solution AB) closed under argon. Solution AB was
then moved to a vented fume hood and magnetically stirred at room
temperature for 2 hours in the dark (reaction vial was wrapped in
aluminum foil). Solution AB was diluted with ethanol (100 mL)
yielding solvent composition of ethanol/chloroform (10:1) (Solution
C). Solution C was diafiltered against 29 dia-volumes (1.5 L) of
binary solvent mixture of ethanol/chloroform (9:1 by volume) at a
flow rate of 800 mL/min and a 30-40 psi back pressure applied by
appropriately adjusting a 1/4 turn ball valve. Under these UFDF
conditions, the permeate flow rate is approximately 5 mL/min.
Solvent was removed from the retentate solution (solution CR) by
rotary evaporation at 35.degree. C. and subsequently vacuum dried
at room temperature of 18 hours to obtain crude product (770 mg,
70%). Crude product was dissolved in 7.7 mL of chloroform (100
mg/mL, solution D). Solution D was added drop-wise to 150 ml of
ether/hexane (70/30) precipitant while stirring. The precipitated
product was recovered by filtration using 0.2 micron PTFE membrane
filter in a Millipore vacuum filtration apparatus. Purified
product, HO-PEG-lys-urea-glu (protected) (white powder, 700 mg,
91%) was obtained by vacuum drying at room temperature for 24 hours
to remove residual solvents.
Example 2
Characterization of HO-PEG-lys-urea-glu (Protected) to Determine
the mol % End Functionalization with of PEG with lys-urea-glu
Targeting Agent and its Molar Mass
[0133] Identity of HO-PEG-lys-urea-glu (protected) was ascertained
by 1H NMR in D.sub.2O using the following instrument parameters:
NMR field strength=400 MHz; Number of scans=128; Pulse delay=5 sec;
Pulse width=7 .mu.sec. NMR peak assignments are as follows: Allyl
sp.sup.2 CH (3H multiplet, 5.9-6.03 ppm), allyl sp.sup.2 CH.sub.2
(6H, two overlapping doublets, 5.27-5.4 ppm), allyl sp.sup.3
CH.sub.2 (6H, two overlapping doublets, 4.6-4.7 ppm), PEG terminal
CH.sub.2--OH (2H triplet, 3.85-3.92 ppm), PEG ethylene oxide
CH.sub.2 (ca. 408H based on M.sub.n=5 KDa, singlet 3.7 ppm), PEG
terminal CH.sub.2 epsilon to CONH-lys-urea-glu (2H triplet,
3.5-3.55 ppm), lysine CH.sub.2 epsilon C (2H two triplets,
3.05-3.14 ppm and 3.15-3.22 ppm), PEG terminal CH.sub.2 alpha to
CONH-lys-urea-glu and PEG terminal CH.sub.2 alpha to COOH of
uncoupled PEG (2H multiplet, 2.15-2.3 ppm), glutamic acid CH.sub.2
gamma C (two 2H overlapping triplets 2.5-2.6 ppm), glutamic acid
CH.sub.2 beta C (two 1H multiplets, 1.8-2.1 ppm), lysine CH.sub.2
beta C (1H multiplet, 1.67-1.8 ppm). The overlapping multiplets
between 1.3 and 1.67 ppm were assigned to (a) lysine CH.sub.2 beta
C (1H), (b) lysine CH.sub.2 gamma C (2H), (c) lysine CH.sub.2 delta
C (2H), (d) PEG terminal CH.sub.2's beta and delta to
CONH-lys-urea-glu (2H each), (e) PEG terminal CH.sub.2 gamma to
CONH-lys-urea-glu (2H). Lys-urea-glu incorporation into
HO-PEG-lys-urea-glu (protected) product was estimated using the
ratio the ligand multiplet between 1.95-2.1 ppm and PEG multiplet
between 2.15-2.3 ppm yielding 82% incorporation of lys-urea-glu
(protected) into HO-PEG-lys-urea-glu (protected) conjugate.
[0134] See FIG. 1 for proton NMR spectra of HO-PEG-lys-urea-glu
(protected), lot numbers 11-189-1 and 11-176-1.
[0135] Molecular weight of HO-PEG-lys-urea-glu (protected) was
determined by Size Exclusion Chromatography using Refractive Index
detection and chloroform as elution solvent. Column
Temperature=30.degree. C., RI Detector temperature=35.degree. C.,
Sample concentration=10 mg/mL, Injection volume=10 uL. Number and
Weight Average Molecular Weight (M.sub.n and M.sub.w) were obtained
relative to narrow disperse Poly(ethylene glycol) standards from
Polymer Standards Service USA (Warwick, R.I.). A fourth order
polynomial fit (R.sup.2=0.99957; Standard Error=0.02) yielded
Mn=5200 Da; Mw=5700 Da; Mw/Mn=1.1 for HO-PEG-lys-urea-glu
(protected).
[0136] See FIG. 2 for Size Exclusion Chromatograms (SEC) of
HO-PEG-lys-urea-glu (protected), lot numbers 11-189-1 (labeled
44-44-6 for SEC analysis) and 11-176-1.
Example 3
Synthesis of PLA-PEG-lys-urea-glu (Protected) by Ring Opening
Polymerization of D,L-Lactide Using HO-PEG-lys-urea-glu (Protected)
as Macroinitiator and Tin(II) 2-ethylhexanoate as Polymerization
Catalyst
[0137] Materials used in the synthesis of PLA-PEG-lys-urea-glu
(protected) included HO-PEG-lys-urea-glu (protected), synthesized
as described in Example 1; Tin (II) 2-ethylhexanoate (95%)
(Sn(Oct).sub.2, Sigma-Aldrich); D,L-lactide (.gtoreq.99.5%,
Altasorb, Piedmont, S.C.); Hexane, anhydrous (95% DriSolv, EMD) in
polymerization reaction and Hexane (Chromasolv., .gtoreq.95%,
Sigma) for work-up precipitation; Diethyl ether (.gtoreq.99.0%,
Sigma);
[0138] Instrumentation used for In-process characterization and
reaction/work-up of PLA-PEG-lys-urea-glu (protected) included a
Bruker 400 MHz Nuclear Magnetic Resonance Spectrometer for Proton
NMR Spectroscopic Analysis at Spectral Data Services, Champaign,
Ill.; Vacuum atmospheres dry nitrogen glove box (<1 ppm water);
12-place parallel reaction carusel (Brinkmann-Hiedolph) equipped
with a multi-position stir plate and solid state temperature
controller (IKA multi-stir plate and temperature controller with
feed-back loop; Schlenk reaction tubes equipped with a Teflon stop
cock side arm. Edwards RV5 Vacuum Pump and a VWR Oven for Vacuum
Drying; Rotary Evaporator (Buchi); Waters Size Exclusion
Chromatography system equipped with Refractive Index Detector and
Waters HR1, HR3 and HR4 columns (7.8.times.300 mm, Molecular Weight
Range 100-5000 Da, 500-30,000 Da, 5000-600,000 Da, respectively).
Number and Weight Average Molar Mass was determined using
Poly(styrene) calibration standards from Shodex Standards,
(Kawasaki, Japan) of Peak Molar Mass (Mp=1200, 3900, 12800, 31,400,
55,100, and 197,000 Da).
[0139] Experimental procedure: All reagents (HO-PEG-lys-urea-glu
(protected), Sn(Oct).sub.2, d,l-lactide, anhydrous hexane) were
transferred to a dry argon glove box and subsequently manipulated
under dry nitrogen at room temperature. HO-PEG-lys-urea-glu (200
mg, 4.times.10.sup.-2 mmol) and lactide (800 mg, 5.55 mmol) were
weighed into reaction tube (containing a suitably size magnetic
stir bar) and septum sealed under dry argon (mixture A).
Sn(Oct).sub.2 (250 mg) was weighed into a 6 mL glass vial and
dissolved in anhydrous hexane (2.5 mL) to yield a 100 mg/mL
solution (B) and septum sealed under dry nitrogen. Mixture A was
removed to a vented fume hood and dried under a steady stream of
dry argon for 1 hour at room temperature using argon gas manifold
equipped with a 8 inch needles as argon gas inlets. Schlenk
reaction tube side arms were connected to a silicone oil bubbler to
serve as argon gas outlet. The Schlenk tubes were subsequently
sealed by stopping the inlet argon flow from the argon gas manifold
and the side arm gas outlet through the schlenk tube side arm using
the Teflon stop cork. Sealed Schlenk tubes were placed in a
130.degree. C. silicone oil bath and magnetically stirrer to obtain
a clear colorless lactide monomer melt (about 10 minutes). Solution
B (80 uL) was introduced via a 500 uL syringe via the septum seal.
The reaction mixture (C) was allowed to stir at 130.degree. C. for
16 hours. Reaction mixture C was cooled to room temperature,
Schlenk tube opened in air and polymer pellet dissolved in 5 mL
dichloromethane by vortexing for 1 hour at room temperature
(solution D). Solution D was transferred to a 20 mL glass vial and
Schlenk tube rinsed with 2.times.2.5 mL portion of fresh
dichloromethane. The washing were combined with solution D. This
slightly turbid solution was filtered using 0.45 micron PTFE
syringe filters to obtain a clear colorless solution (Solution E).
Solution E was added drop-wise to diethyl ether/hexane (70/30) (200
mL) while stirring at room temperature. The cloudy precipitate was
allowed to stir at room temperature for 2 hours to allow polymer to
coagulate and settle to the bottom of the beaker. The clear
supernatant was decanted and the tacky polymer product (700 mg,
70%) was transferred to glass vial using spatula and subsequently
dried under vacuum at room temperature for 18 hours.
Example 4
Characterization of PLA-PEG-lys-urea-glu (Protected) to Determine
the mol % End Functionalization with of PEG with lys-urea-glu
Targeting Agent and its Molar Mass
[0140] Identity of PLA-PEG-lys-urea-glu (protected) was ascertained
by .sup.1H NMR in chloroform-d using the following instrument
parameters: NMR field strength=400 MHz; Number of scans=128; Pulse
delay=5 sec; Pulse width=7 .mu.sec. NMR peak assignments are as
follows: Lactide methine CH (1H multiplet, 5.1-5.3 ppm), Lactide
methyl CH.sub.3 (3H multiplet, 1.45-1.65), PEG ethylene oxide
CH.sub.2 (ca. 408H based on M.sub.n=5 KDa, singlet 3.55-3.8), allyl
sp.sup.2 CH (3H multiplet, 5.8-6.0 ppm). The number average molar
mass (Mn) of PLA-PEG-lys-urea-glu (protected) was determined by
proton NMR spectroscopy using PEG to poly(d,l-lactide) ratio.
Comparison of the intensities of the allyl sp.sup.2 CH (3H
multiplet, 5.8-6.0 ppm) peak and the lactide methine CH (1H
multiplet, 5.1-5.3 ppm) peak yielded M.sub.n (PLA-PEG-lys-urea-glu
(protected)=20,100 Da assuming PEG M.sub.n=5 Kg/mol.
[0141] See FIG. 3 for proton NMR spectra of PLA-PEG-lys-urea-glu
(protected), lot numbers 11-187-1, 11-188-1 and 11-198-1.
[0142] Molecular weight of PLA-PEG-lys-urea-glu (protected) was
also determined by Size Exclusion Chromatography using Refractive
Index detection and chloroform as elution solvent. Column
Temperature=30.degree. C., RI Detector temperature=35.degree. C.,
Sample concentration=10 mg/mL, Injection volume=10 uL. M.sub.n and
M.sub.w were obtained relative to narrow disperse Polystyrene
Standards (Shodex Standards, Kawasaki, Japan). A fourth order
polynomial fit (R.sup.2=0.999977; Standard Error=0.009) yielded
M.sub.n=20700 Da; M.sub.w=25400 Da; Mw/Mn=1.23 for
PLA-PEG-PLA-PEG-lys-urea-glu (protected).
[0143] See FIG. 4 for Size Exclusion Chromatograms (SEC) of
HO-PEG-lys-urea-glu (protected), lot numbers 11-189-1 (labeled
44-44-6 for SEC analysis) and 11-176-1.
Example 5
Synthesis of PLA-PEG-lys-urea-glu by Removal of the Allyl
Protecting Groups of PLA-PEG-lys-urea-glu (Protected) Using
Tetrakis(triphenylphosphine) Palladium (0)
[0144] Materials used for the synthesis of PLA-PEG-lys-urea-glu
included PLA-PEG-lys-urea-glu (protected), synthesized as described
in example 3;
[0145] Tetrakis(triphenylphosphene)palladium(0)(99%) (Pd-tetrakis,
Sigma); Morpholine (99.5%, Sigma); Dichloromethane (Anhydrous,
.gtoreq.99.8%, Sigma); Diethyl ether (.gtoreq.99.0%, Sigma); Hexane
(Chromasolv., .gtoreq.95%, Sigma).
[0146] Instrumentation and equipment used for work-up procedures
and in-process characterization included a Bruker 400 MHz Nuclear
Magnetic Resonance Spectrometer for Proton NMR Spectroscopic
Analysis; Edwards RV5 Vacuum Pump and a VWR Oven for Vacuum Drying;
Rotary Evaporator (Buchi).
[0147] Experimental procedure: PLA-PEG-lys-urea-glu (protected)
(1000 mg, 4.76.times.10.sup.-2 mmol) was dissolved in anhydrous
dichloromethane (4 mL) under ambient air (Solution A). Pd-tetrakis
(55 mg, 4.76.times.10.sup.-2 mmol) was dissolved in anhydrous
dichloromethane (4 mL) also under ambient air (Solution B).
Morpholine (41.5 mg or 45.2 uL, 0.476 mmol) was added to solution
A, this was immediately followed by the addition of Solution B to
Solution A. Dichloromethane (2 mL) was used to rinse the Solution B
vial and this rinse solution was combined with Solution A to ensure
quantitative transfer of the Pd-tetrakis to the reaction mixture
(C). Mixture C was allowed to stir closed and in the dark (reaction
vial was wrapped in aluminum foil) at room temperature for 2 hours.
Reaction mixture C was opened in air and added dropwise into
diethyl ether/hexane (70/30, (v/v)) (200 mL) at room temp while
stirring. The resulting polymer suspension was allowed to stir at
room temperature for 2 hours allowing it to coagulate and settle to
the bottom of the beaker. The clear supernatant was decanted off
and the tacky polymer was transferred using spatula to a glass
vial. PLA-PEG-lys-urea-glu (800 mg, 80%) product was dried under
vacuum for 18 hours at room temperature.
Example 6
Palladium Removal from PLA-PEG-lys-urea-glu Using Trimercapto
Triazide Functional Palladium Scavenging Resin
[0148] PLA-PEG-lys-urea-glu (1050 mg) was dissolved in
dichloromethane (30 mL) to yield a 35 mg/mL solution. Palladium
scavenging resin (TMT, 5 g in 60 mL column) was solvated with
dichloromethane (10 mL) and PLA-PEG-lys-urea-glu solution (30 mL)
added. Eluant was collected under gravity and column rinsed with
additional dichloromethane (40 mL). Main and rinse eluant fractions
combined and solvent removed by rotary evaporation to recover
polymer. Polymer was subsequently dissolved in dichloromethane
(10.5 mL, 100 mg/mL solution) and added dropwise to 70/30
ether/hexane (210 mL). The resulting polymer suspension was allowed
to stir at room temperature for 2 hours allowing it to coagulate
and settle to the bottom of the beaker. The clear supernatant was
decanted off and the tacky PLA-PEG-lys-urea-glu polymer was
transferred using spatula to a glass vial (940 mg, 90%).
Example 7
Characterization of PLA-PEG-lys-urea-glu to Determine the
Efficiency of Deprotection, Molar Mass and Residual Palladium
Content
[0149] The identity of PLA-PEG-lys-urea-glu was ascertained by
.sup.1H NMR in chloroform-d using the following instrument
parameters: NMR field strength=400 MHz; Number of scans=128; Pulse
delay=5 sec; Pulse width=7 .mu.sec. NMR peak assignments are as
follows: Lactide methine CH (1H multiplet, 5.1-5.3 ppm), Lactide
methyl CH.sub.3 (3H multiplet, 1.45-1.7), PEG ethylene oxide
CH.sub.2 (ca. 408H based on M.sub.n=5 KDa, singlet 3.55-3.8),
residual allyl sp.sup.2 CH (3H multiplet, 5.8-6.0 ppm).
[0150] The efficiency of deprotection reaction was also determined
by NMR as 95% (allyl removal). This was calculated using the ratio
of the intensity of the residual allyl peak in the product
PLA-PEG-lys-urea-glu spectrum to that of the corresponding peak in
the starting material (PLA-PEG-lys-urea-glu (protected))
spectrum.
[0151] The number average molar mass (MO of PLA-PEG-lys-urea-glu
was estimated by NMR using PEG to poly(d,l-lactide) ratio.
Comparison of the intensities of the PEG ethylene oxide CH.sub.2
(408H, singlet 3.55-3.8) peak and the lactide methine CH (1H
multiplet, 5.1-5.3 ppm) peak yielded M.sub.n
(PLA-PEG-lys-urea-glu)=21,400 Da assuming PEG M.sub.n=5 Kg/mol.
[0152] Palladium content in product PLA-PEG-lys-urea-glu was
determined by ICP Spectrometry and found <5 ppm.
[0153] The molar mass of PLA-PEG-lys-urea-glu was estimated by
measurement of inherent viscometry (IV) in dimethylsulfoxide and
chloroform (0.309 dL/g and 0.198 dL/g, respectively). The I.V. of
the protected precursor PLA-PEG-lys-urea-glu (protected) was
determined in dimethylsulfoxide (0.217 dL/g). A minor drop in
viscosity from 0.217 dL to 0.198 dL/g was observed and may be
attributed to differences in polymer solvent interactions as a
result of the more polar nature of lys-urea-glu relative to the
allyl protected precursor.
[0154] See FIG. 5 for proton NMR spectra of crude (prior to
palladium removal) PLA-PEG-lys-urea-glu, lot numbers 11-187-1,
11-188-1 and 11-198-1.
[0155] See FIG. 6 for proton NMR spectra of purified (after
palladium removal) PLA-PEG-lys-urea-glu, lot number 44-49-1.
[0156] See FIG. 7 for Inherent viscosity of purified (after
palladium removal) PLA-PEG-lys-urea-glu, lot number 44-49-1 and the
protected precursor PLA-PEG-lys-urea-glu (protected)
[0157] See FIG. 8 for Palladium Content in crude (prior to
palladium removal) PLA-PEG-lys-urea-glu lot number 44-48-1 (sample
code 11-204-1) and purified (after palladium removal)
PLA-PEG-lys-urea-glu lot number 44-49-1 (sample code 11-204-2)
determined by ICP Spectrometry.
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