U.S. patent application number 13/978713 was filed with the patent office on 2014-10-23 for hydrophobic molecule-induced branched polymer aggregates and their use.
This patent application is currently assigned to ANP Technologies, Inc.. The applicant listed for this patent is Jing Pan, Dujie Qin, Ray Yin, Yubei Zhang. Invention is credited to Jing Pan, Dujie Qin, Ray Yin, Yubei Zhang.
Application Number | 20140314664 13/978713 |
Document ID | / |
Family ID | 46457985 |
Filed Date | 2014-10-23 |
United States Patent
Application |
20140314664 |
Kind Code |
A1 |
Qin; Dujie ; et al. |
October 23, 2014 |
Hydrophobic Molecule-Induced Branched Polymer Aggregates And Their
Use
Abstract
Symmetrically and asymmetrically branched homopolymers are
modified at the surface level with functional groups that enable
forming aggregates with water insoluble or poorly water soluble
pharmaceutically active agents (PAA). The aggregates formed are
specifically induced by interaction of PAA and homopolymer and are
different from aggregates that are formed by the polymer alone in
the absence of the PAA or by the PAA alone in the absence of the
polymer. Such aggregates can be used to improve drug solubility,
stability, delivery and efficacy.
Inventors: |
Qin; Dujie; (Wilmington,
DE) ; Yin; Ray; (Newark, DE) ; Pan; Jing;
(Newark, DE) ; Zhang; Yubei; (Hockessin,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Qin; Dujie
Yin; Ray
Pan; Jing
Zhang; Yubei |
Wilmington
Newark
Newark
Hockessin |
DE
DE
DE
DE |
US
US
US
US |
|
|
Assignee: |
ANP Technologies, Inc.
Newark
DE
|
Family ID: |
46457985 |
Appl. No.: |
13/978713 |
Filed: |
January 6, 2012 |
PCT Filed: |
January 6, 2012 |
PCT NO: |
PCT/US12/20524 |
371 Date: |
October 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61431042 |
Jan 9, 2011 |
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61500633 |
Jun 24, 2011 |
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61502793 |
Jun 29, 2011 |
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Current U.S.
Class: |
424/1.37 ;
424/178.1; 424/184.1; 424/649; 424/9.1; 424/9.3; 514/283;
514/449 |
Current CPC
Class: |
A61K 9/14 20130101; A61P
31/00 20180101; A61K 51/06 20130101; A61K 45/06 20130101; A61K
47/59 20170801; A61P 35/00 20180101; A61K 31/4375 20130101; A61K
9/5146 20130101; A61K 31/337 20130101; A61K 9/19 20130101; A61K
47/58 20170801; A61K 49/12 20130101; A61K 47/6803 20170801; A61K
49/0004 20130101; A61K 33/24 20130101; A61K 9/513 20130101; A61K
31/4745 20130101; A61K 47/34 20130101 |
Class at
Publication: |
424/1.37 ;
424/649; 424/178.1; 424/184.1; 424/9.1; 424/9.3; 514/449;
514/283 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 47/48 20060101 A61K047/48; A61K 49/00 20060101
A61K049/00; A61K 45/06 20060101 A61K045/06; A61K 51/06 20060101
A61K051/06; A61K 31/337 20060101 A61K031/337; A61K 31/4375 20060101
A61K031/4375; A61K 33/24 20060101 A61K033/24; A61K 49/12 20060101
A61K049/12 |
Claims
1. An aggregate comprising a) a branched homopolymer, wherein said
homopolymer is modified with a surface group and wherein said
surface modified branched homopolymer comprises symmetrically
branched polymers, asymmetrically branched polymer or a combination
thereof, and b) a water insoluble or poorly water soluble
pharmaceutically active agent (PAA), wherein the size of said
aggregate is different from the size of aggregates formed of said
surface modified branched homopolymer alone.
2. The aggregate of claim 1, wherein said surface modified branched
homopolymer comprises a hydrophobic surface group.
3. The aggregate of claim 1, wherein said PAA is associated with a
surface group of said branched polymer.
4. The aggregate of claim 2, wherein said surface modified branched
polymer comprises a polyoxazoline, a poly(2-substituted oxazoline),
a polyethyleneglycol, a polyethyleneoxide, a polyacrylamide, a
polyphosphate, a polyvinylpyrrolidone, a polyvinyl alcohol, a
polyethyleneimine, a polypropyleneimine, a polyamidoamine or a
combination thereof.
5. The aggregate of claim 2, wherein said surface modified branched
polymer comprises an aliphatic and/or saturated or unsaturated
hydrocarbons comprising from 1 to about 22 carbons, aromatic
hydrocarbons, polyethylene polymers, polystyrene polymers,
perfluoropolymers, polydimethylsiloxanes, polyacrylates,
polymethylmethacrylates or a combination thereof.
6. The aggregate of claim 1, wherein said water insoluble or poorly
water soluble PAA comprises an antiinfective agent or an
antineoplastic agent.
7. The aggregate of claim 1, wherein said water insoluble or poorly
water soluble PAA comprises paclitaxel, docetaxel, taxotere,
vinblastine, vincristine, vindesine, vinorelbine, irinotecan,
topotecan, camptothecin, camptothecin derivatives (such as,
irinotecan, topotecan etc.), doxorubin, cisplatin, carboplatin,
oxaliplatin, satraplatin, dolargin, loperamide, tubocurarine,
ibuprofen, diazepam, naproxen, carbamazepine, griseofulvin,
nifedipine, phytosterol, omeprazol, domperidone, zidovudine,
amphotericin B, chlormethine, chlorambucil, busulfan, thiotepa,
cyclophosphamide, estramustine, ifosfamide, meclilorethamine,
melphalan, uramustine, lonuistine, streptozotocin, dacarbazine,
procarbazine, temozolamide,
(SP-4-3)-(cis)-aminedichloro-[2-methylpyridine]-platinum (II),
methotrexate, permetrexed, raltitrexed, trimetrexate, cladribine,
chlorodeoxyadenosine, clofarabine, fludarabine, mercaptopurine,
pentostatin, thioguanine, azacitidine, capecitabine, cytarabine,
edatrexate, floxuridine, 5 fluorouracil, gemcitabine,
troxacitabine, bleomycin, dactinomycin, adriamycin, actinomycin,
mithramycin, mitomycin, mitoxantrone, porfiromycin, daunorubicin,
epirubicin, idarubicin, valrubicin, phenesterine, tamoxifen,
piposulfancamptothesin, amsacrine, etoposide, teniposide,
fluoxymesterone, testolactone, bicalutamide, cyproterone,
flutamide, nilutamide, aminoglutethimide, anastrozole, exemestane,
formestane, letrozole, dexamethasone, prednisone,
diethylstilbestrol, fulvestrant, raloxifene, toremifene, buserelin,
goserelin, leuprolide, triptorelin, medroxyprogesterone acetate,
megestrol acetate, levothyroxine, liothyronine, altretamine,
levamisole, mitotane, octreotide, procarbazine, suramin,
thalidomide, methoxsalen, sodium porfimer, bortezomib, erlotinib
hydrochloride, gefitinib, imatinib mesylate, semaxanib, adapalene,
bexarotene, trans-retinoic acid, 9-cis-retinoic acid and
N-(4-hydroxyphenyl) retinamide or a combination thereof.
8. The aggregate of claim 1, further comprising a targeting moiety
covalently linked to said homopolymer, wherein said moiety
comprises antibody, antigen-binding portion thereof, antigen, cell
receptor, cell receptor ligand or lectin ligand.
9. The aggregate of claim 1, further comprising a contrast
reagent.
10. The aggregate of claim 4, wherein said poly(2-substituted
oxazoline) comprises poly(2-methyloxazoline,
poly(2-ethyloxazoline), poly(2-propyloxazoline) or
poly(2-butyloxazoline).
11. The aggregate of claim 9, wherein said contrast agent comprises
a magnetic resonance imaging contrast agent.
12. The aggregate of claim 9, wherein said contrast agent comprises
a radionuclide.
Description
[0001] The instant application claims benefit to U.S. Ser. No.
61/431,042 filed 9 Jan. 2011, U.S. Ser. No. 61/500,633 filed 24
Jun. 2011, and U.S. Ser. No. 61/502,793 filed 29 Jun. 2011, the
content of each of which is incorporated herein by reference in
entirety.
FIELD
[0002] The present disclosure relates to a surface modified
branched polymer (MBP), which can either be a surface modified
symmetrically branched polymer (SBP) or a surface modified
asymmetrically branched polymer (ABP), which on exposure to a water
insoluble or poorly water soluble molecule, such as, a drug, forms
a composite nanoparticle or nanoaggregate, wherein the drug is
dispersed or deposited primarily at the surface of the structures
where hydrophobic portions or sites are located. The particles or
aggregates of interest are stable, for example, can be desiccated
and rehydrated. The nanoparticles or nanoaggregates can range from
about 50 nm to about 500 nm in diameter depending, in part, on the
drug to polymer ratio, the drug, the polymer, the solvent(s) used,
amount of the homopolymer and amount of the drug. Hydrophobic,
electrostatic, metal-ligand interactions, hydrogen bonding and
other molecular interactions may be involved in the spontaneous
interactions between the water insoluble or poorly water soluble
molecule and the homopolymer to form aggregates. The particles or
aggregates of interest have a controlled release profile and thus
find utility, for example, as a carrier for the controlled release
of pharmacologically active agents, drugs and the like in a host,
for providing a supplement, nutrient or requirement; for treating
any of a variety of disorders; and the like.
BACKGROUND
Symmetrically Branched Polymers
[0003] A new class of polymers called dendritic polymers, including
Starburst dendrimers (or Dense Star polymers) and Combburst
dendrigrafts (or hyper comb branched polymers), recently was
developed and studied for various industrial applications. Those
polymers often possess: (a) a well defined core molecule, (b) at
least two concentric dendritic layers (generations) with
symmetrical (equal length) branches and branch junctures, and (c)
exterior surface groups, such as, polyamidoamine (PAMAM)-based
branched polymers and dendrimers described in U.S. Pat. Nos.
4,435,548; 4,507,466; 4,568,737; 4,587,329; 5,338,532; 5,527,524;
and 5,714,166. Other examples include polyethyleneimine (PEI)
dendrimers, such as those disclosed in U.S. Pat. No. 4,631,337;
polypropyleneimine (PPI) dendrimers, such as those disclosed in
U.S. Pat. Nos. 5,530,092; 5,610,268; and 5,698,662; Frechet-type
polyether and polyester dendrimers, core shell tectodendrimers and
others, as described, for example, in "Dendritic Molecules", edited
by Newkome et al., VCH Weinheim, 1996; "Dendrimers and Other
Dendritic Polymers", edited by Frechet & Tomalia, John Wiley
& Sons, Ltd., 2001; and U.S. Pat. No. 7,754,500.
[0004] Combburst dendrigrafts are constructed with a core molecule
and concentric layers with symmetrical branches through a stepwise
synthetic method. In contrast to dendrimers, Combburst dendrigrafts
or polymers are generated with monodisperse linear polymeric
building blocks (U.S. Pat. Nos. 5,773,527; 5,631,329 and
5,919,442). Moreover, the branch pattern is different from that of
dendrimers. For example, Combburst dendrigrafts form branch
junctures along the polymeric backbones (chain branches), while
Starburst dendrimers often branch at the termini (terminal
branches). Due to the living polymerization techniques used, the
molecular weight distributions (M.sub.w/M.sub.n) of those polymers
(core and branches) often are narrow. Thus, Combburst dendrigrafts
produced through a graft-on-graft process are well defined with
M.sub.w/M.sub.n ratios often less than about 1.
[0005] SBP's, such as dendrimers, are predominantly produced by
repetitive protecting and deprotecting procedures through either a
divergent or a convergent synthetic approach. Since dendrimers
utilize small molecules as building blocks for the cores and the
branches, the molecular weight distribution of the dendrimers often
is defined. In the case of lower generations, a single molecular
weight dendrimer often is obtained.
[0006] In addition to dendrimers and dendrigrafts, other SBP's
include symmetrical star shaped or comb shaped polymers, such as,
symmetrical star shaped or comb shaped polyethyleneoxide (PEO),
polyethyleneglycol (PEG), PEI, PPI, polyoxazoline (PDX),
polymethyloxazoline (PMOX), polyethyloxazoline (PEOX), polystyrene,
polymethylmethacrylate, polydimethylsiloxane or a combination
thereof.
Asymmetrically Branched Polymers
[0007] Unlike SBP's, asymmetrically branched polymers (ABP),
particularly asymmetrically branched dendrimers or regular ABP
(reg-ABP), often possess a core, controlled and well defined
asymmetrical (unequal length) branches and asymmetrical branch
junctures as described in U.S. Pat. Nos. 4,289,872; 4,360,646; and
4,410,688.
[0008] On the other hand, a random ABP (ran-ABP) possesses: a) no
core, b) functional groups both at the exterior and in the
interior, c) random/variable branch lengths and patterns (i.e.,
termini and chain branches), and d) unevenly distributed interior
void spaces.
[0009] The synthesis and mechanisms of ran-ABPs, such as, made of
PEI, was reported by Jones et al., J. Org. Chem. 9, 125 (1944),
Jones et al., J. Org. Chem. 30, 1994 (1965) and Dick et al., J.
Macromol. Sci. Chem., A4 (6), 1301-1314, (1970)). Ran-ABP, such as
those made of PDX, i.e., poly(2-methyloxazoline) and
poly(2-ethyloxazoline), were reported by Litt (J. Macromol. Sci.
Chem. A9(5), 703-727 (1975)) and Warakomski (J. Polym. Sci. Polym.
Chem. 28, 3551 (1990)). The synthesis of ran-ABP's often can
involve a one-pot divergent or a one-pot convergent method.
Homopolymers
[0010] A homopolymer can relate to a polymer or to a polymer
backbone composed of the same repeat unit, that is, the homopolymer
is generated from the same monomer (e.g., polyethyleneimine
dendrimers, polyamidoamine dendrimers or polyoxazoline dendrimers).
The monomer can be a simple compound or a complex or an assemblage
of compounds where the assemblage or complex is the repeat unit in
the homopolymer. Thus, if an assemblage is composed of three
compounds, A, B and C; the complex can be depicted as ABC. A
polymer composed of (ABC)-(ABC)-(ABC) . . . is a homopolymer for
the purposes of the instant disclosure. The homopolymer may be
linear or branched. Thus, in the case of a randomly branched PEI,
although there are branches of different length and branches occur
randomly, that molecule is a homopolymer for the purposes of the
instant disclosure because that branched polymer is composed of a
single monomer, ethyleneimine or aziridine. Also, one or more of
the monomer or complex monomer components can be modified,
substituted, derivatized and so on, for example, modified to carry
a functional group. Such molecules are homopolymers for the
purposes of the instant disclosure as the backbone is composed of a
single simple or complex monomer.
Poorly Water Soluble Drugs
[0011] Small molecule drug candidates and drugs, as well as
biological molecules, which can be modified for particular purposes
or to have particular properties, may be poorly soluble or
insoluble in water. Generally, the need for hydrophilicity for a
molecule to survive in circulation or in tissue spaces can
constrain the use of pharmacologically active hydrophobic drug
candidates or drugs. Hence, development of effective formulations
for poorly water soluble pharmaceutically active agents (PAA) is
important in drug development and use. Current solutions include
improving drug solubility or reducing drug particle size by, for
example, chemical modification or physical formulation.
[0012] Chemical modification methods often involve converting the
drug, e.g., by using a salt form, hydrating or attaching various
water soluble functional groups, such as, amino/imino, hydroxyl, or
carboxyl containing groups; water soluble polymers, such as, PEG or
PEO, and the like to the original drug molecule to enhance water
solubility.
[0013] Physical formulation can include using a cosolvent and/or a
surfactant to dissolve a poorly soluble drug; involving a lipid or
a liposome-based nanoemulsion or microemulsion; melting drug and
polymer without any solvents at elevated temperatures; using a
complexing agent (e.g., an inorganic salt, coordination metals
(e.g., hexamine cobalt (III) chloride), chelates (e.g., EDTA, EGTA
etc.), metal-olefins or metallocenes (e.g., Ferrocene), inclusion
compounds (e.g., cyclodextrins, choleic acid etc.) or molecular
complexes); as well as solid dispersion in a carrier, such as,
e.g., acids, such as, citric acid, tartaric acid, succinic acid,
HCl etc.), sugars (e.g., dextrose, sorbitol, sucrose, maltose,
galactose, xylitol etc.), polymeric materials (e.g.,
polyvinylpyrrolidone, PEG-400, PEG-1000, PEG-4000, PEG-6000,
carboxymethyl cellulose, hydroxypropyl cellulose, guar gums,
xanthan gums, sodium alginates, methyl celluloses, HPMC,
cyclodextrins and their derivatives, galactomannans, surfactants
(e.g., polyoxyethylene stearate, a poloxamer, a deoxycholic acid, a
Tween, a Span, a Gelucire, a vitamin E TPGS etc.), and the like
(e.g., pentaerythritol, urea, urethane, hydroxyalkyl xanthenes
etc.).
[0014] Other known strategies include drug particle size reduction,
for example, micronization, which can use a milling technique, such
as, use of a jet mill or a rotor stator colloid mill to reduce
particle size; increase dissolution rate with increased surface
area; nanosuspension, which is a submicron colloidal dispersion of
pure particles of drugs, which can be stabilized by surfactants;
homogenization, which often involves conventional homogenizers,
sonicators and high shear fluid processors; wet milling, where the
active drug is fragmented in the presence of surfactant by milling
or by spraying drug dissolved in a volatile organic solvent into a
heated aqueous solution; using supercritical fluids; polymorph
changes; using eutectic mixtures; using self microemulsifying drug
delivery systems etc.
[0015] However, those treatments may compromise pharmacologic
activity.
[0016] While drugs often can be delivered through various routes,
including oral, intrathecal, rectal, intranasal, subdermal,
subdural, intramuscular, transdermal, topical, inhalation,
injection and so on, intravenous drug delivery allows rapid and
direct equilibration of the drug in the circulation, that can
enable effective local concentration. A stable and controlled drug
release formulation not only can avoid excessively high serum
levels just after dosing but also can allow gradual release of the
drug in the intravascular compartment.
[0017] Microparticles larger than 7 .mu.m are generally cleared
from the circulation by the "blood filtering organs," such as, the
spleen, lungs and liver. Therefore, smaller nanoparticles, e.g.,
50-500 nm, often possess longer blood circulation times.
[0018] Examples of pharmaceutically active agents (PAA), such as,
drugs, include, but are not limited to, chlormethine, chlorambucil,
busulfan, thiotepa, cyclophosphamide, estramustine, ifosfamide,
meclilorethamine, melphalan, uramustine, lonuistine,
streptozotocin, dacarbazine, procarbazine, temozolamide, cisplatin,
carboplatin, oxaliplatin, satraplatin,
(SP-4-3)-(cis)-aminedichloro-[2-methylpyridine]-platinum (II),
methotrexate, permetrexed, raltitrexed, trimetrexate, camptothecin,
camptothecin derivatives (such as, irinotecan, topotecan etc.),
cladribine, chlorodeoxyadenosine, clofarabine, fludarabine,
mercaptopurine, pentostatin, thioguanine, azacitidine,
capecitabine, cytarabine, edatrexate, floxuridine, 5-fluorouracil,
gemcitabine, troxacitabine, bleomycin, dactinomycin, adriamycin,
actinomycin, mithramycin, mitomycin, mitoxantrone, porfiromycin,
daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin,
idarubicin, valrubicin, phenesterine, tamoxifen,
piposulfancamptothesin, L-asparaginase, PEG-L-asparaginase,
paclitaxel, docetaxel, taxotere, vinblastine, vincristine,
vindesine, vinorelbine, irinotecan, topotecan, amsacrine,
etoposide, teniposide, fluoxymesterone, testolactone, bicalutamide,
cyproterone, flutamide, nilutamide, aminoglutethimide, anastrozole,
exemestane, formestane, letrozole, dexamethasone, prednisone,
diethylstilbestrol, fulvestrant, raloxifene, toremifene, buserelin,
goserelin, leuprolide, triptorelin, medroxyprogesterone acetate,
megestrol acetate, levothyroxine, liothyronine, altretamine,
arsenic trioxide, gallium nitrate, hydroxyurea, levamisole,
mitotane, octreotide, procarbazine, suramin, thalidomide,
methoxsalen, sodium porfimer, bortezomib, erlotinib hydrochloride,
gefitinib, imatinib mesylate, semaxanib, adapalene, bexarotene,
trans-retinoic acid, 9-cis-retinoic acid and N-(4-hydroxyphenyl)
retinamide, alemtuzumab, bevacizumab, cetuximab, ibritumomab
tiuxetan, rituximab, trastuzumab, gemtuzumab ozogamicin,
tositumomab, interferon-.alpha.2a, interferon-.alpha. and so on,
and derivatives and modifications thereof, so long as the drug, or
derivative thereof, is poorly soluble or insoluble in water. Some
of the molecules above are modified to be more soluble in water.
For the purposes of the instant disclosure, such molecules can be
modified or altered to remove such modifications resulting in a
pharmaceutically active or biologically active molecule which is
less hydrophilic and more hydrophobic, that is, poorly water
soluble or water insoluble.
[0019] Thus, PAA's that are water insoluble or poorly water
soluble, or those which are sensitive to acid environments
generally cannot be conventionally administered (e.g., by
intravenous injection or oral administration). In some
circumstances, parenteral administration of such pharmaceuticals
can be achieved by emulsification of oil-solubilized drug with an
aqueous liquid (such as normal saline), often in the presence of
surfactants or emulsifiers to produce an emulsion for
administration.
[0020] For example, paclitaxel is a water insoluble drug.
Paclitaxel is sold as Taxol.RTM. by Bristol-Myers Squibb.
Paclitaxel is derived from the Pacific Yew tree, Taxus brevifolia
(Wan et al., J. Am. Chem. Soc. 93:2325 (1971). Taxanes, including
paclitaxel and docetaxel (also sold as Taxotere.RTM.) are used to
treat various cancers, including, breast, ovarian and lung cancers,
as well as colon, and head and neck cancers, etc.
[0021] However, the poor aqueous solubility of paclitaxel has
hampered the widespread use thereof. Currently, Taxol.RTM. and
generics thereof are formulated using a 1:1 solution of
ethanol:Cremaphor.RTM. (polyethyoxylated castor oil) to solubilize
the drug. The presence of Cremaphor.RTM. has been linked to severe
hypersensitivity reactions and consequently requires medication of
patients with corticosteroids (e.g., dexamethasone) and
antihistamines.
[0022] Alternatively, conjugated paclitaxel, for example,
Abraxane.RTM., which is produced by mixing paclitaxel with human
serum albumin, has eliminated the need for corticosteroids and
antihistamine injections. However, Abraxane.RTM. generates
undesirable side effects, such as, severe cardiovascular events,
including chest pain, cardiac arrest, supraventricular tachycardia,
edema, thrombosis, pulmonary thromboembolism, pulmonary emboli,
hypertension etc, which prevents patients with high cardiovascular
risk from using the drug.
Delivery of Poorly Water Soluble Drugs with Surface Modified
Branched Polymers
[0023] Although branched polymers, including SBP's and ABP's, have
been used for drug delivery, those attempts are primarily focused
on the chemical attachment of the drug to the polymer, or physical
encapsulation of such drugs in the interior through unimolecular
encapsulation (U.S. Pat. Nos. 5,773,527; 5,631,329; 5,919,442; and
6,716,450).
[0024] For example, dendrimers and dendrigrafts are believed to
physically entrap bioactive molecules using unimolecular
encapsulation approaches, as described in U.S. Pat. Nos. 5,338,532;
5,527,524; and 5,714,166 for dense star polymers, and U.S. Pat. No.
5,919,442 for hyper comb branched polymers. Similarly, the
unimolecular encapsulation of various drugs using SBP's to form a,
"dendrimer box," was reported in Tomalia et al., Angew. Chem. Int.
Ed. Engl., 1990, 29, 138, and in "Dendrimers and Other Dendritic
Polymers", edited by Frechet & Tomalia, John Wiley & Sons,
Ltd., 2001, 387-424.
[0025] Branched core shell polymers with a hydrophobic core and a
hydrophilic shell may be used to entrap a poorly water soluble drug
through molecular encapsulation. Randomly branched and
hyperbranched core shell structures with a hydrophilic core and a
hydrophobic shell have also been used to carry a drug through
unimolecular encapsulation and pre-formed nanomicelles (U.S. Pat.
No. 6,716,450 and Liu et al., Biomaterials 2010, 10, 1334-1341).
However, those unimolecular and pre-formed micelle structures are
generated in the absence of a drug.
[0026] Block copolymers, such as miktoarm polymers (i.e., Y
shape/AB.sub.2 type star polymers) and linear (A)-dendritic (B)
block copolymers, were observed to form sterocomplexes with
paclitaxel (Nederberg et al., Biomacromolecules 2009, 10, 1460-1468
and Luo et al., Bioconjugate Chem. 2010, 21, 1216). Those block
copolymers closely resemble traditional lipid or AB-type linear
block copolymers, which are well known surfactants used for the
generation of micelles.
[0027] However, such branched block copolymers are difficult to
make and thus, are not suitable for mass production.
[0028] There are no descriptions of modifying branched
homopolymers, which on exposure to a poorly soluble or water
insoluble drug, spontaneously form stable aggregates which are
suitable for controlled drug delivery.
SUMMARY
[0029] In one aspect, the present disclosure is directed to use of
modified branched polymers (MBP) to increase the solubility of
water insoluble or poorly water soluble pharmaceutically active
agents (PAA), such as, drugs. Such MBP's can include both
symmetrically and asymmetrically branched polymers.
[0030] In another aspect of the disclosure, the symmetrically
branched polymer (SBP) has regular symmetrical branches within the
polymer. In another aspect of the disclosure, the asymmetrically
branched polymer (ABP) has either random or regular, asymmetrical
branches. The random ABP can also have a mixture of terminal and
chain branching patterns.
[0031] In another aspect of the disclosure, both ABP's and SBP's
can be modified further with at least one molecule or group capable
of forming additional branches at a given time so that new material
properties can be achieved, wherein additional functional groups
may be further attached. All of the modified polymers can be
defined as modified symmetrically or asymmetrically branched
polymers.
[0032] In another aspect of the disclosure, the unmodified and
modified branched polymers either can be produced by a divergent or
a convergent method, and either a stepwise or a one-step synthetic
process can be used.
[0033] In another aspect of the disclosure, the SBP includes, but
is not limited to, polyamidoamine dendrimers; polyethyleneimine
dendrimers; polypropyleneimine dendrimers; polyether dendrimers;
polyester dendrimers; comb branched/star branched polymers, such
as, polyamidoamine, polyethyleneoxide (PEO), polyethyleneglycol
(PEG), polymethyloxazoline, polyethyloxazoline,
polymethylmethacrylate (PMA), polystyrene, polybutadiene,
polyisoprene and polydimethylsiloxane; comb branched dendrigrafts,
such as, polyethyloxazoline, polymethyloxazoline,
polyethyleneimine, polyamidoamine; and so on.
[0034] In a further aspect of the disclosure, the SBP can have an
interior void space, while the ABP can have unevenly distributed
void spaces.
[0035] In another aspect of the disclosure, a hybrid branched
polymer comprising the aforementioned SBP's, such as, dendrimers or
dendrigrafts, and ABP's, such as, regular and randomly branched
polymers, as well as star branched and comb branched polymers, or
combination thereof, can also be used for the generation of said
drug-induced aggregates or nanoparticles of interest.
[0036] In another aspect of the disclosure, the branched polymers
are modified with functional groups, such as, but not limited to,
NH.sub.2, NHR, NR.sub.2, NR.sub.3.sup.+, COOR, COOH, COO.sup.-, OH,
C(O)R, C(O)NH.sub.2, C(O)NHR or C(O)NR.sub.2, wherein R can be any
aliphatic group, aromatic group or combination thereof; an
aliphatic group (e.g., a hydrocarbon chain), which can be branched,
can contain one or more double and/or triple bonds and/or may be
substituted; an aromatic group, which may contain a plurality of
rings, which may be fused or separated, the rings may be of varying
size and/or may contain substituents; perfluorocarbon chains;
saccharides and/or polysaccharides, which may be of varying ring
sizes, the rings may contain a heteroatom, such as a sulfur or a
nitrogen atom, may be substituted, may contain more than one
species of saccharide, may be branched and/or may be substituted;
polyethylene glycols; and the like.
[0037] The molecular weight of the MBP's can range from about 500
to over 5,000,000; from about 500 to about 1,000,000; from about
1,000 to about 500,000; or from about 2,000 to about 100,000.
[0038] In another aspect of the disclosure, the surface of the
symmetrically and asymmetrically polymers is modified so that the
physical properties of the surface groups will be more compatible
with a PAA of interest, thus making the PAA more miscible with the
surface group region/domain of the MBP's.
[0039] In an embodiment, the modification of branched polymers is
with hydrophobic functional groups, such as, aliphatic chains
(e.g., hydrocarbon chains comprising 1 to about 22 carbons, whether
linear or branched), aromatic structures (e.g. containing one or
more aromatic rings, which may be fused) or combinations
thereof.
[0040] In contrast to known drug carriers, the PAA's of the instant
disclosure are not physically entrapped within said branched
polymer structures. Instead, the PAA either can be located at or
dispersed in the domains/regions containing surface functional
groups of each branched polymer.
[0041] The resulting structures of interest optionally can be
preserved, for example, by lyophilization or other form of
desiccation, which may further stabilize the structures of
interest. Once redissolved in water or a buffer, nanoparticles with
sizes ranging from about 50 to about 500 nm in diameter can be
obtained.
[0042] The presence of multiple, often functionalized branches
enables the formation of intramolecular and intermolecular
crosslinks, which may stabilize the PAA-containing nanoparticles.
On dilution, said physical aggregate or nanoparticle deconstructs
releasing drug at a controlled rate.
[0043] In another aspect of the disclosure, the branched polymer
can comprise targeting moieties/groups including, but not limited
to, an antibody or antigen-binding portion thereof, antigen,
cognate carbohydrates (e.g., sialic acid), a cell surface receptor
ligand, a moiety bound by a cell surface receptor, a moiety that
binds a cell surface saccharide, an extracellular matrix ligand, a
cytosolic receptor ligand, a growth factor, a cytokine, an
incretin, a hormone, a lectin, a lectin ligand, such as, a
galactose, a galactose derivative, an N-acetylgalactosamine, a
mannose, a mannose derivative and the like, a vitamin, such as, a
folate or a biotin, avidin, streptavidin, neutravidin, DNA, RNA
etc. Such targeted nanoparticles release drug at the preferred
treatment locations, and therefore, enhance local effective
concentrations and can minimize undesired side effects.
[0044] In another aspect of the disclosure, a targeting
moiety/group and a functional group, including, hydrophobic,
hydrophilic and/or ionic functional groups, are attached to the
branched polymer prior to the formation of the composite
nanoparticle for targeted drug delivery.
[0045] In another aspect of the disclosure, a diagnostic agent,
such as, a contrast reagent, also can be carried by said structures
of interest, for example, for in vivo imaging. In an embodiment,
the diagnostic agent is one which is poorly soluble or insoluble in
water, thereby negating a need for a drug to form a structure of
interest.
[0046] In some embodiments, an imaging agent is one comprising a
metal or is paramagnetic, e.g., magnetic resonance imaging
materials, which can be deposited or entrapped within said branched
polymer of said nanocomposite-based particle.
[0047] In yet another aspect of the disclosure, the diagnostic
material containing nanoparticles can further comprise a targeting
moiety/group, which allows such nanoparticle to target specific
locations for diagnosis.
[0048] In other embodiments, a structure of interest comprises a
second or more PAA's. The second or more PAA's may or may not be
poorly soluble or insoluble in water.
[0049] In another aspect of disclosure, the nanoparticle can carry
plural similar PAA's or can carry PAA's of different function or
activity, such as, types of drugs to form a combination or cocktail
therapy. Such drugs may include, but are not limited to, small
molecule drugs, inorganic drugs and biological molecule-based
drugs, such as peptides, proteins, antibodies or antigen-binding
portions thereof, enzymes, vaccines and so on, for the treatment of
various diseases or general use, such as, for cosmetics and over
the counter products.
[0050] In another aspect of the disclosure, the nanoparticle-based
drug formulations also can be used in drug discovery and
development, where various therapeutic formulations can be screened
and tested rapidly.
[0051] Additional features and advantages of the present disclosure
are described in, and will be apparent from, the following Detailed
Description and the attached Figures.
BRIEF DESCRIPTION OF THE FIGURES
[0052] The following description of the figures and the respective
drawings are non-limiting examples that depict various embodiments
that exemplify the present disclosure.
[0053] FIG. 1 depicts SBP's including a dendrimer, a star shaped
polymer, a dendrigraft and a comb shaped polymer. All have a core,
whether globular or linear.
[0054] FIG. 2 depicts a chemical structure of symmetrically
branched PPI dendrimers.
[0055] FIG. 3 depicts chemical modification reactions of
symmetrically branched PPI dendrimers. The numbers, 8, 16, 32 and
so on indicate the number of reactive groups at the surface of the
dendrimer.
[0056] FIGS. 4A and 4B depict random (A) and regular (B) ABP's with
asymmetrical branch junctures and patterns.
[0057] FIG. 5 depicts a chemical structure of a random
asymmetrically branched PEI homopolymer.
[0058] FIGS. 6A and 6B depict synthetic schemes. FIG. 6A presents
chemical modification reactions of random asymmetrically branched
PEI homopolymers. FIG. 6B depicts a one-pot synthesis of
hydrophobically modified, randomly branched poly(2-ethyloxazoline)
with a primary amino group at the focal point of the polymer. The
initiator/surface group (I) is the brominated hydrocarbon. The
reaction opens the oxazoline ring.
[0059] FIG. 7 illustrates a drug loaded in or at the surface domain
or region of the branched polymer (SBP's and ABP's). In this and
other figures, R indicates a surface group and a solid circle
depicts a drug of interest.
[0060] FIG. 8 illustrates one type of composite-based nanoparticles
containing both drug molecules and branched polymers.
[0061] FIG. 9 illustrates an insoluble or poorly water soluble drug
that is loaded at hydrophobic surface groups of branched polymers
(SBP's and ABP's). In this and other figures, a thin, wavy line
depicts a hydrophobic surface group.
[0062] FIG. 10 illustrates various drug-containing nanoparticles
also carrying at least one targeting group or moiety, such as, an
antibody, depicted herein and in other figures as a, "Y."
[0063] FIG. 11 illustrates drug-containing nanoparticles carrying
both magnetic imaging contrast agents and a targeting moiety or
group, such as an antibody. In this and other Figures, M denotes an
imaging material, such as, a magnetic resonance imaging contrast
agent.
[0064] FIG. 12 illustrates drug-containing nanoparticles carrying
both radioactive (Rad) agents and a targeting moiety or group, such
as, an antibody.
[0065] FIG. 13 shows the size comparison of polymer-only and
polymer-drug aggregates with the polymer concentration at 25 mg/mL
and the drug concentration at 5 mg/mL in saline. The polymer is a
hydrophobically-modified, randomly-branched PEOX and the drug is
paclitaxel.
[0066] FIG. 14 shows the size comparison of polymer-only and
polymer-drug aggregates with the polymer concentration at 2.5 mg/mL
and the drug concentration at 0.5 mg/mL in saline. The polymer is a
hydrophobically-modified, randomly-branched PEOX and the drug is
paclitaxel.
[0067] FIG. 15 shows the size comparison of polymer-only and
polymer-drug aggregates with the polymer concentration at 250
.mu.g/mL and the drug concentration at 50 .mu.g/mL in saline. The
polymer is a hydrophobically-modified, randomly-branched PEOX and
the drug is paclitaxel.
[0068] FIG. 16 shows the size comparison of polymer-only and
polymer-drug aggregates with the polymer concentration at 25
.mu.g/mL and the drug concentration at 5 .mu.g/mL in saline. The
polymer is a hydrophobically-modified, randomly-branched PEOX and
the drug is paclitaxel.
[0069] FIG. 17 summarizes data comparing cytotoxicity of a drug and
a drug aggregate. MCF-7 human breast cancer cells were exposed to
various concentrations of each and survivability was
determined.
[0070] FIG. 18 summarizes data comparing cytotoxicity of a drug and
a drug aggregate. H460 human epithelial lung cancer cells were
exposed to various concentrations of each and survivability was
determined.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0071] The drug solubility in this disclosure is defined as,
relative to parts of solvent required to solubilize for one part of
drug, <30 (soluble), 30-100 (poorly soluble) and >100
(insoluble).
[0072] For the purposes of the instant disclosure, the words, such
as, "about," "substantially" and the like are defined as a range of
values no greater than 20% from the stated value or figure.
"Homopolymer," is as described hereinabove.
Drugs of Interest
[0073] The PAA described in this disclosure includes any
chemical/small molecule-based drug, inorganic-based drug,
biological/large molecule-based drug, modifications and/or
derivatives thereof, and combinations thereof, wherein the drug is
poorly soluble or insoluble in water. Hence, a drug of interest can
be a small molecule, a salt thereof in which the molecule is
modified to be water insoluble or poorly water soluble or can be a
biological molecule which is modified to be water insoluble or
poorly water soluble.
[0074] Chemical/small molecule drugs can include any substantially
poorly soluble or water insoluble pharmacologically active agents
contemplated for use in the practice of the present disclosure
include PAA's, drugs, imaging agents, diagnostic agents, agents of
nutritional value, supplements, vitamins, lifestyle chemicals and
the like. Some of those may need to be converted to a less water
soluble form, for example, changing the PAA from a salt to a
non-salt form or from a charged to a non-charged molecule.
[0075] Suitable examples of PAA's include drugs which are poorly
soluble or insoluble in water, which include, growth agents, AIDS
adjunct agents, alcohol abuse preparations, such as, agents for
treating dependence or withdrawal, Alzheimer's Disease treatments,
Amyotrophic Lateral Sclerosis treatments, analgesics, anesthetics,
anticonvulsants, antidiabetic agents, antidotes, antifibrosis
therapies, antihistamines, anti-infective agents, such as,
antibiotics, antivirals, antifungals, amebicides, antihelmintics,
antimalarials, leprostatics and so on, antineoplastics,
antiparkinsonian agents, antirheumatic agents, appetite stimulants,
biological response modifiers, biologicals, blood modifiers, such
as, anticoagulants, colony stimulating factors, hemostatics, plasma
extenders, thrombin inhibitors and so on, bone metabolism
regulators, cardioprotective agents, cardiovascular agents, such
as, adrenergic blockers, adrenergic stimulators, ACE inhibitors,
antiarrhythmics, antilipemic agents, calcium channel blockers,
diuretics, vasopressors and so on, CNS stimulants, cholinesterase
inhibitors, contraceptives, fertility treatments, ovulation
stimulators, cystic fibrosis managements agents, detoxifying
agents, diagnostics, dietary supplements, dopamine receptor
agonists, endometriosis management agents, enzymes, erectile
dysfunction treatments, foot care products, GI agents, such as
antacids, antidiarrheals, antiemetics, antiflatulants, bowel
evacuants, digestive enzymes, histamine receptor agonists,
laxatives, proton pump inhibitors, prostaglandins and so on,
Gaucher's Disease treatments, gout treatments, homeopathic
remedies, skin treatments, vitamins, nutrients, hormones,
hypercalcemia management treatments, hypocalcemia management
treatments, immunomodulators, immunosuppressants, levocarnitine
deficiency treatments, mast cell stabilizers, migraine treatments,
motion sickness products, such as, benadryl and phenergan,
decongestants, antihistamines, cough suppressants, multiple
sclerosis treatments, muscle relaxants, nasal preparations, such
as, antiinflammatories, smoking cessation aids, appetite
suppressants, nucleoside analogs, obesity managements, ophthalmic
preparations, such as, antibiotics, antiglaucoma agents, artificial
tears, lubricants and so on, sexual aids, lubricants, osteoporosis
treatments, otic preparations, such as, antiinfectives and
cerumenolytics, minerals, oxytocics, parasympatholytics,
parasympathomimetics, patent ductus arteriosus agents, phosphate
binders, porphyria agents, prostaglandins, psychotherapeutic
agents, radiopaque agents, respiratory agents, such as,
antiinflammatories, antitussives, bronchodilators, decongestants,
expectorants, leukotrienes antagonists, surfactants and so on, salt
substitutes, sedatives, hypnotics, skin and mucous membrane
preparations, such as, acne treatments, anorectal treatments, such
as, hemorrhoid treatments and enemas, antiperspirants,
antipruritics, antipsoriatic agents, antiseborrheic agents, burn
treatments, cleansing agents, depigmenting agents, emollients, hair
growth retardants, hair growth stimulators, keratolytics, hair
problem treatments, mouth and throat problem treatments, shampoos,
photosensitizing agents, wart treatments, wound care treatments and
so on, over the counter pharmaceutics and products, such as,
deodorants, Tourette's Syndrome agents, tremor treatments, urinary
tract agents, such as, acidifiers, alkalinizers, antispasmodics,
benign prostatic hyperplasia treatments, calcium oxalate stone
preventors, enuresis management agents and so on, vaginal
preparations, such as, antiinfectives, hormones and so on,
vasodilators, vertigo treatments, Wilson's Disease treatments and
so on.
[0076] Listed herein are drugs of interest as well as forms of
drugs which may be modified, for example, as salts. For the
purposes of the invention, any such ionized or hydrophilic forms
are modified as known in the art to remove such functional groups,
modifications and the like to yield non-modified or other forms of
drugs which are poorly soluble or not soluble in water. Examples of
pharmaceutically active agents, drugs and the like include those
listed herein and, for example, analgesics/antipyretics (e.g.,
aspirin, acetaminophen, ibuprofen, naproxen sodium, buprenorphine
hydrochloride, propoxyphene hydrochloride, propoxyphene napsylate,
meperidine hydrochloride, hydromorphone hydrochloride, morphine
sulfate, oxycodone hydrochloride, codeine phosphate, dihydrocodeine
bitartrate, pentazocine hydrochloride, hydrocodone bitartrate,
levorphanol tartrate, diflunisal, trolamine salicylate, nalbuphine
hydrochloride, mefenamic acid, butorphanol tartrate, choline
salicylate, butalbital, phenyltoloxamine citrate, diphenhydramine
citrate, methotrimeprazine, cinnamedrine hydrochloride, meprobamate
and the like); anesthetics (e.g., cyclopropane, enflurane,
halothane, isoflurane, methoxyflurane, nitrous oxide, propofol and
the like); antiasthmatics (e.g., azelastine, ketotifen, traxanox,
amlexanox, cromolyn, ibudilast, montelukast, nedocromil, oxatomide,
pranlukast, seratrodast, suplatast tosylate, tiaramide,
zafirlukast, zileuton, beclomethasone, budesonide, dexamethasone,
flunisolide, triamcinolone acetonide and the like); antibiotics
(e.g., neomycin, streptomycin, chloramphenicol, cephalosporin,
ampicillin, penicillin, tetracycline and the like); antidepressants
(e.g., nefopam, oxypertine, doxepin hydrochloride, amoxapine,
trazodone hydrochloride, amitriptyline hydrochloride, maprotiline
hydrochloride, phenelzine sulfate, desipramine hydrochloride,
nortriptyline hydrochloride, tranylcypromine sulfate, fluoxetine
hydrochloride, doxepin hydrochloride, imipramine hydrochloride,
imipramine pamoate, nortriptyline, amitriptyline hydrochloride,
isocarboxazid, trimipramine maleate, protriptyline hydrochloride
and the like); antidiabetics (e.g., biguanides, hormones,
sulfonylurea derivatives, and the like); antifungal agents (e.g.,
griseofulvin, ketoconazole, amphotericin B, nystatin, candicidin
and the like); antihypertensive agents (e.g., propanolol,
propafenone, oxyprenolol, nifedipine, reserpine, trimethaphan
camsylate, phenoxybenzamine hydrochloride, pargyline hydrochloride,
deserpidine, diazoxide, guanethidine monosulfate, minoxidil,
rescinnamine, sodium nitroprusside, rauwolfia serpentina,
alseroxylon, phentolamine mesylate, reserpine and the like);
anti-inflammatories (e.g., non-steroidal compounds, such as,
indomethacin, naproxen, ibuprofen, ramifenazone, piroxicam and so
on, and steroidal compounds, such as, cortisone, dexamethasone,
fluazacort, hydrocortisone, prednisolone, prednisone and the like);
antineoplastics (e.g., adriamycin, cyclophosphamide, actinomycin,
bleomycin, daunorubicin, doxorubicin, epirubicin, mitomycin,
methotrexate, fluorouracil, carboplatin, carmustine (BCNU),
methyl-CCNU, cisplatin, etoposide, interferons, camptothecin and
derivatives thereof, phenesterine, Taxol and derivatives thereof,
taxotere and derivatives thereof, vinblastine, vincristine,
tamoxifen, etoposide, piposulfan and the like); antianxiety agents
(e.g., lorazepam, buspirone hydrochloride, prazepam,
chlordiazepoxide hydrochloride, oxazepam, clorazepate dipotassium,
diazepam, hydroxyzine pamoate, hydroxyzine hydrochloride,
alprazolam, droperidol, halazepam, chlormezanone, dantrolene and
the like); immunosuppressive agents (e.g., cyclosporine,
azathioprine, mizoribine, FK506 (tacrolimus) and the like);
antimigraine agents (e.g., ergotamine tartrate, propanolol
hydrochloride, isometheptene mucate, dichloralphenazone and the
like); sedatives/hypnotics (e.g., barbiturates (e.g.,
pentobarbital, pentobarbital sodium, secobarbital sodium and the
like) or benzodiazapines (e.g., flurazepam hydrochloride,
triazolam, tomazeparm, midazolam hydrochloride and the like);
antianginal agents (e.g., .beta.-adrenergic blockers, calcium
channel blockers (e.g., nifedipine, diltiazem hydrochloride and the
like) and nitrates (e.g., nitroglycerin, isosorbide dinitrate,
pentaerythritol tetranitrate, erythrityl tetranitrate and the
like)); antipsychotic agents (e.g., haloperidol, loxapine
succinate, loxapine hydrochloride, thioridazine, thioridazine
hydrochloride, thiothixene, fluphenazine hydrochloride,
fluphenazine decanoate, fluphenazine enanthate, trifluoperazine
hydrochloride, chlorpromazine hydrochloride, perphenazine, lithium
citrate, prochlorperazine and the like); antimanic agents (e.g.,
lithium carbonate); antiarrhythmics (e.g., bretylium tosylate,
esmolol hydrochloride, verapamil hydrochloride, amiodarone,
encamide hydrochloride, digoxin, digitoxin, mexiletine
hydrochloride, disopyramide phosphate, procainamide hydrochloride,
quinidine sulfate, quinidine gluconate, quinidine
polygalacturonate, flecamide acetate, tocamide hydrochloride,
lidocaine hydrochloride and the like); antiarthritic agents (e.g.,
phenylbutazone, sulindac, penicillamine, salsalate, piroxicam,
azathioprine, indomethacin, meclofenamate sodium, gold sodium
thiomalate, ketoprofen, auranofin, aurothioglucose, tolmetin sodium
and the like); antigout agents (e.g., colchicine, allopurinol and
the like); anticoagulants (e.g., heparin, heparin sodium, warfarin
sodium and the like); thrombolytic agents (e.g., urokinase,
streptokinase, altoplase and the like); antifibrinolytic agents
(e.g., aminocaproic acid); hemorheologic agents (e.g.,
pentoxifylline); antiplatelet agents (e.g., aspirin, empirin,
ascriptin and the like); anticonvulsants (e.g., valproic acid,
divalproate sodium, phenyloin, phenyloin sodium, clonazepam,
primidone, phenobarbitol, phenobarbitol sodium, carbamazepine,
amobarbital sodium, methsuximide, metharbital, mephobarbital,
mephenyloin, phensuximide, paramethadione, ethotoin, phenacemide,
secobarbitol sodium, clorazepate dipotassium, trimethadione and the
like); antiparkinson agents (e.g., ethosuximide and the like);
antihistamines/antipruritics (e.g., hydroxyzine hydrochloride,
diphenhydramine hydrochloride, chlorpheniramine maleate,
brompheniramine maleate, cyproheptadine hydrochloride, terfenadine,
clemastine fumarate, triprolidine hydrochloride, carbinoxamine
maleate, diphenylpyraline hydrochloride, phenindamine tartrate,
azatadine maleate, tripelennamine hydrochloride,
dexchlorpheniramine maleate, methdilazine hydrochloride,
trimprazine tartrate and the like); agents useful for calcium
regulation (e.g., calcitonin, parathyroid hormone and the like);
antibacterial agents (e.g., amikacin sulfate, aztreonam,
chloramphenicol, chloramphenicol palmitate, chloramphenicol sodium
succinate, ciprofloxacin hydrochloride, clindamycin hydrochloride,
clindamycin palmitate, clindamycin phosphate, metronidazole,
metronidazole hydrochloride, gentamicin sulfate, lincomycin
hydrochloride, tobramycin sulfate, vancomycin hydrochloride,
polymyxin B sulfate, colistimethate sodium, colistin sulfate and
the like); antiviral agents (e.g., interferon .gamma., zidovudine,
amantadine hydrochloride, ribavirin, acyclovir and the like);
antimicrobials (e.g., cephalosporins (e.g., cefazolin sodium,
cephradine, cefaclor, cephapirin sodium, ceftizoxime sodium,
cefoperazone sodium, cefotetan disodium, cefutoxime azotil,
cefotaxime sodium, cefadroxil monohydrate, ceftazidime, cephalexin,
cephalothin sodium, cephalexin hydrochloride monohydrate,
cefamandole nafate, cefoxitin sodium, cefonicid sodium, ceforanide,
ceftriaxone sodium, ceftazidime, cefadroxil, cephradine, cefuroxime
sodium and the like), penicillins (e.g., ampicillin, amoxicillin,
penicillin G benzathine, cyclacillin, ampicillin sodium, penicillin
G K, penicillin V K, piperacillin sodium, oxacillin sodium,
bacampicillin hydrochloride, cloxacillin sodium, ticarcillin
disodium, azlocillin sodium, carbenicillin indanyl sodium,
penicillin G procaine, methicillin sodium, nafcillin sodium and the
like), erythromycins (e.g., erythromycin ethylsuccinate,
erythromycin, erythromycin estolate, erythromycin lactobionate,
erythromycin stearate, erythromycin ethylsuccinate and the like),
tetracyclines (e.g., tetracycline hydrochloride, doxycycline
hyclate, minocycline hydrochloride and the like), and the like);
anti-infectives (e.g., GM-CSF); bronchodilators (e.g.,
sympathomimetics (e.g., epinephrine hydrochloride, metaproterenol
sulfate, terbutaline sulfate, isoetharine, isoetharine mesylate,
isoetharine hydrochloride, albuterol sulfate, albuterol,
bitolterol, mesylate isoproterenol hydrochloride, terbutaline
sulfate, epinephrine bitartrate, metaproterenol sulfate,
epinephrine, epinephrine bitartrate); anticholinergic agents (e.g.,
ipratropium bromide); xanthines (e.g., aminophylline, dyphylline,
metaproterenol sulfate, aminophylline); mast cell stabilizers
(e.g., cromolyn sodium); inhalant corticosteroids (e.g.,
flunisolide, beclomethasone dipropionate monohydrate and the like),
salbutamol, beclomethasone dipropionate (BDP), ipratropium bromide,
budesonide, ketotifen, salmeterol, xinafoate, terbutaline sulfate,
triamcinolone, theophylline, nedocromil sodium, metaproterenol
sulfate, albuterol, flunisolide and the like); hormones (e.g.,
androgens (e.g., danazol, testosterone cypionate, fluoxymesterone,
ethyltostosterone, testosterone enanthate, methyltestosterone,
fluoxymesterone, testosterone cypionate and the like); estrogens
(e.g., estradiol, estropipate, conjugated estrogens and the like),
progestins (e.g., methoxyprogesterone acetate, norethindrone
acetate and the like), corticosteroids (e.g., triamcinolone,
betamethasone, betamethasone sodium phosphate, dexamethasone,
dexamethasone sodium phosphate, dexamethasone acetate, prednisone,
methylprednisolone acetate suspension, triamcinolone acetonide,
methylprednisolone, prednisolone sodium phosphate
methylprednisolone sodium succinate, hydrocortisone sodium
succinate, methylprednisolone sodium succinate, triamcinolone
hexacatonide, hydrocortisone, hydrocortisone cypionate,
prednisolone, fluorocortisone acetate, paramethasone acetate,
prednisolone tebulate, prednisolone acetate, prednisolone sodium
phosphate, hydrocortisone sodium succinate and the like), thyroid
hormones (e.g., levothyroxine sodium); and the like); and the like;
hypoglycemic agents (e.g., human insulin, purified beef insulin,
purified pork insulin, glyburide, chlorpropamide, glipizide,
tolbutamide, tolazamide and the like); hypolipidemic agents (e.g.,
clofibrate, dextrothyroxine sodium, probucol, lovastatin, niacin
and the like); proteins (e.g., DNase, alginase, superoxide
dismutase, lipase and the like); nucleic acids (e.g., sense or
anti-sense nucleic acids encoding any therapeutically useful
protein, including any of the proteins described herein and the
like); agents useful for erythropoiesis (e.g., erythropoietin);
antiulcer or antireflux agents (e.g., famotidine, cimetidine,
ranitidine hydrochloride and the like); antinauseants or
antiemetics (e.g., meclizine hydrochloride, nabilone,
prochlorperazine, dimenhydrinate, promethazine hydrochloride,
thiethylperazine, scopolamine and the like); oil-soluble vitamins
(e.g., vitamins A, D, E, K and the like); and as well as other
drugs such as mitotane, visadine, halonitrosoureas, anthrocyclines,
ellipticine and the like.
[0077] Examples of diagnostic agents contemplated for use in the
practice of the present disclosure include, but are not limited to,
for example, magnetic resonance imaging contrast agents (e.g.,
various metal ions, such as, gadolinium based compounds for
functional MRI, fluorocarbons, lipid soluble paramagnetic compounds
and the like), ultrasound contrast agents, radiocontrast agents,
such as, conventional radionuclides, such as, iodine, copper,
fluorine, gallium, thallium and the like, which may be complexed
with a carrier (e.g., iodo-octanes, halocarbons, renografin and the
like), as well as other diagnostic agents which cannot readily be
delivered without some physical and/or chemical modification to
accommodate the substantially water insoluble nature thereof.
Metals and radionuclides can be carried or bound to a protein,
lipid, nucleic acid, chelator, such as a small molecule, or
combinations thereof.
[0078] Examples of agents of nutritional or lifestyle value
contemplated for use in the practice of the present disclosure
include amino acids, sugars, lipids, proteins, carbohydrates, oils,
such as, fish oil, fat-soluble vitamins (e.g., vitamins A, D, E, K
and the like), minerals, supplements, fats, emollients, tanning
agents, moisturizers and the like, or combinations thereof.
Nanocomposite or Nanoaggregate
[0079] A nanocomposite is a physical mixture of two or more
materials or components (e.g., polymer and PAA molecules). In the
instant disclosure, such a mixture could contain different
nanoscopic phases or domains formed between the PAA and branched
homopolymer molecule in either solid or liquid states.
Nanocomposite can include a combination of a bulk matrix (e.g.,
branched homopolymers and PAA's) and nanodimensional phase(s),
which may exhibit different properties due to dissimilarities of
structure and chemistry (e.g., the domain formed by the PAA and the
surface groups of branched polymer, as well as the domains formed
by the interior of the branched polymers). Since the solubility of
the domains/phases may be different, on dissolving the
nanocomposite in an aqueous solution, one of the phases may
dissolve faster than the other or others, resulting in a gradual
breakdown of the composite aggregate resulting in a graded and
controlled release of the composite components and optionally,
reformation of one or more of the components into a novel form,
such as, a new aggregate.
[0080] The size of the aggregates described in the disclosure
ranges from between about 10 to about 500 nm in diameter, or from
about 30 nm to about 300 nm in diameter. Aggregates may exhibit
size-related properties that differ significantly from those
observed for microparticles or bulk materials.
[0081] SBP's are depicted in FIG. 1, with symmetric branches,
wherein all the homopolymers of interest possess a core and exhibit
symmetric branch junctures consisting either of terminal or chain
branches throughout the homopolymer. The functional groups are
present predominantly at the exterior.
[0082] The modified SBP's can be obtained, for example, through
chemically linking functional groups on, for example, symmetrically
branched PAMAM or PPI dendrimers, commercially available from
Aldrich, polyether dendrimers, polyester dendrimers, comb
branched/star branched polymers, such as, those containing PEO,
PEG, PMOX or PEOX, polystyrene, and comb branched dendrigrafts,
such as, those containing PEOX, PMOX or PEI.
[0083] The synthetic procedures for making such SBP's/dendrimers
are known (see, for example, "Dendrimers and Other Dendritic
Polymers," Frechet & Tomalia, eds., John Wiley & Sons,
Ltd., 2001) using commercially available reagents (for example,
various generations of PPI dendrimers, FIG. 2) or a number of SBP's
are commercially available. The synthesis of comb branched and
combburst polymers is known (see, for example, U.S. Pat. Nos.
5,773,527; 5,631,329; and 5,919,442).
[0084] The higher branching densities of SBP's render the polymers
molecularly compact with a well defined interior void space, which
makes such molecules suitable as a carrier for entities, such as,
reporters or PAA's entrapped or encased therein.
[0085] The surface modifications can enhance the properties and
uses of the resulting modified SBP's. For example, with suitable
modification, a water insoluble SBP can become water soluble, while
an SBP with a high charge density can be modified to carry very low
or no charge on the polymer or at the polymer surface. On the other
hand, a water soluble SBP can be modified with hydrophobic surface
groups to enhance the ability to solubilize water insoluble or
poorly water soluble drugs at the surface thereof.
[0086] In one embodiment of the instant disclosure, the SBP (for
example, either a symmetrically branched PEI dendrimer, a PPI
dendrimer, a PAMAM dendrimer or a symmetrically branched PEI
dendrigraft) can be modified with different kinds of, for example,
primary amine groups through, for example, Michael addition or an
addition of acrylic esters onto amine groups of the homopolymer.
Thus, for example, through a Michael addition reaction, methyl
acrylate can be introduced onto the primary and/or secondary amino
groups of PEI, PPI and polylysine (PLL) homopolymers. The ester
groups then can be further derivatized, for example, by an
amidation reaction. Thus, for example, such an amidation reaction
with, for example, ethylenediamine, can yield the addition of an
amino group at the terminus of the newly formed branch. Other
modifications to the homopolymer can be made using known
chemistries, for example, as provided in, "Poly(amines) and
Poly(ammonium salts)" in Handbook of Polymer Synthesis (Part A),
Kricheldorf, ed., New York, Marcel Dekker, 1994; and "Dendrimers
and Other Dendritic Polymers," Frechet & Tomalia, eds., John
Wiley & Sons, Ltd., 2001.
[0087] On such addition, a modified SBP, such as, a modified PEI,
PPI, PAMAM dendrimer or PEI dendrigraft, is formed. As an extension
of the SBP, such as PPI and PEI, the resulting modified SBP also is
symmetrically branched. Depending on the solvent environment (i.e.
pH or polarity), the surface functional groups can carry different
charge and/or charge density, and/or hydrophobic groups. The
molecular shape and surface functional group locations (i.e.,
surface functional group back folding) then can be tuned further,
based on those characteristic properties.
[0088] In another embodiment of the disclosure, the modified SBP's
can be produced using any of a variety of synthetic schemes that,
for example, are known to be amenable to reaction with a suitable
site on the homopolymer. Moreover, any of a variety of reagents can
be used in a synthetic scheme of choice to yield any of a variety
of modifications or additions to the homopolymer backbone. Thus,
for example, in the case of the Michael addition reaction to an
amine described above, the addition of any of a variety of
substituents can be used, for example, at the alkylation stage,
using for example, any of a variety of acrylate reagents, such as,
an acrylate comprising a hydrocarbon substituent, such as saturated
or unsaturated hydrocarbons comprising 1 to about 22 carbons, which
my be substituted, aliphatic, aromatic, ringed, saturated at one or
more bonds or a combination thereof. Thus, suitable reactants
include, methyl acrylate, ethyl acrylate, propyl acrylate, butyl
acrylate, pentyl acrylate, hexyl acrylate, heptyl acrylate, octyl
acrylate, nonyl acrylate, decyl acrylate, undecyl acrylate, dodecyl
acrylate and so on, and mixtures thereof. Similarly, at the
amidation stage in the example exemplified above, any of a variety
of amines can be used. For example, ethylenediamine,
monoethanolamine, tris(hydroxymethyl)aminomethane, alkyl amine,
allyl amine, or any amino modified polymer, including those
comprising PEG, PEO, perfluoropolymers, polystyrene, polyethylene,
polydimethylsiloxane, polyacrylate, polymethylmethacrylate and the
like, and mixtures thereof, can be used.
[0089] Such a synthetic strategy would allow not only symmetric
growth of the molecule, where more branches with different chemical
compositions can be introduced, but also the addition of multiple
functional groups at the exterior of the structure. The precursor
homopolymer can be modified, and continuously, using the same or a
different synthetic process until the desired SBP's with
appropriate molecular weight and functional groups are attained. In
addition, the hydrophobic and hydrophilic properties, as well as
charge densities of such polymers, can be tailored to fit specific
application needs using appropriate monomers for constructing the
homopolymer and suitable modification reactions.
[0090] In another embodiment of the disclosure, if a divergent
synthetic procedure is used, the chain end of symmetrically star
branched or comb branched homopolymer, such as, a
poly(2-substituted oxazoline), including, for example,
poly(2-methyloxazoline), poly(2-ethyloxazoline),
poly(2-propyloxazoline) and poly(2-butyloxazoline, etc.), PEI,
PEO/glycol, polyvinylpyrrolidone, polyphosphate, polyvinyl alcohol
or polystyrene, can be modified with another small molecule or
polymer to generate various functional groups at the homopolymeric
chain ends including a primary, secondary or tertiary amine,
carboxylate, hydroxyl, aliphatic (e.g., hydrocarbon chain),
aromatic, fluoroalkyl, aryl, PEG, PEO, acetate, amide and/or ester
groups. Alternatively, various initiators also can be utilized so
that the same type of functional groups can be introduced at the
chain end if a convergent synthetic approach is utilized (Dendritic
Molecules, Newkome et al., eds., VCH, Weinheim, 1996; Dendrimers
and Other Dendritic Polymers, Frechet & Tomalia, eds., John
Wiley & Sons, Ltd., 2001; and J. Macromol. Sci. Chem. A9(5),
pp. 703-727 (1975)).
[0091] ABP's are depicted in FIGS. 4A and 4B with asymmetric
branches, wherein some of the polymers of interest possess no core
and exhibit asymmetrical branch junctures consisting of both chain
and terminal branches throughout the entire homopolymer. The
functional groups often are present both at the exterior and in the
interior. However, when a larger functional group (e.g., a large
hydrophobic or hydrophilic group) is used, the functional groups
often can be attached preferentially and perhaps necessarily at the
exterior of the ABP, for example, possibly due to steric effects.
Therefore, such surface MBP's can be utilized for solubilization of
or aggregate formation with an insoluble or poorly soluble
drug.
[0092] The modified ABP's can be obtained, for example, through
chemically linking functional groups on regular ABP's, such as,
polylysine (e.g., branched PLL), on random ABP's, such as, PEI's
(commercially available from Aldrich, Polysciences, or BASF under
the trade name, Luposal.TM.) or polyoxazolines, which can be
prepared according to the procedure of Litt (J. Macromol. Sci.
Chem. A9(5), pp. 703-727 (1975)). Other ABP's can include, but are
not limited to, polyacrylamides, polyphosphates,
polyvinylpyrrolidones, polyvinyl alcohols, etc.
[0093] A variety of known starting materials can be used. For
making such modified ABP's. Such monomers and polymers are
available commercially in large quantities at modest cost. For
example, one such precursor monomer that can be used to synthesize
a homopolymer of interest is PEI. The synthesis of random
asymmetrically branched PEI's is known (Jones et al., J. Org. Chem.
9, 125 (1944)). PEI's with various molecular weights are available
commercially from different sources, such as, Aldrich, Polysciences
and BASF (under the trade name Luposal.TM.) The random
asymmetrically branched PEI's are produced primarily through
cationic ring opening polymerization of ring strained cyclic imine
monomers, such as aziridines (ethyleneimine) and azetidines
(propyleneimine), with Lewis or Bronsted acids as initiators.
(Dermer et al., "Ethylenediamine and Other Aziridines", Academic
Press, New York, (1969); and Pell, J. Chem. Soc. 71 (1959)). Since
many of the methods are essentially one-pot processes, large
quantities of random ABP's can be readily produced. Randomly
branched poly(2-substituted oxazoline) polymers can be prepared
using the procedure of Litt (J. Macromol. Sci. Chem. A9 (5), pp.
703-727 (1975)).
[0094] The synthetic processes for making ABP's often generate
various branch junctures within the macromolecule. In other words,
a mixture of terminal and chain branch junctures is distributed
throughout the molecular structure. The branching densities of the
random ABP's can be lower, and the molecular structure can be more
open when compared with dendrimers and dendrigrafts. Although the
branch pattern is random, the average ratio of primary, secondary
and tertiary amine groups can be relatively consistent with a ratio
of about 1:2:1, as described by Dick et al., J. Macromol. Sci.
Chem., A4 (6), 1301-1314 (1970) and Lukovkin, Eur. Polym. J. 9, 559
(1973).
[0095] The presence of the branch junctures can make the random
ABP's, such as, asymmetrically branched PEI's, form macromolecules
with a possible spherical, ovoid or similar configuration. Within
the globular structure, there are various sizes of pockets formed
from the imperfect branch junctures at the interior of the
macromolecule. Unlike dendrimers and dendrigrafts where interior
pockets are always located around the center core of the molecule,
the pockets of random ABP's are spread unevenly throughout the
entire molecule. As a result, random ABP's possess both exterior
and unevenly distributed interior functional groups that can be
further reacted with a variety of molecules, thus forming new
macromolecular architectures, a modified random ABP of
interest.
[0096] Although having a core, the functional groups of the regular
ABP are also distributed both at the exterior and in the interior,
which is very similar to the random ABP. One such homopolymer is
PLL, which can be made as described in U.S. Pat. Nos. 4,289,872;
4,360,646; and 4,410,688. Such homopolymers also can be modified in
a manner similar as that for random ABPs, as taught herein, and as
known in the art.
[0097] In one embodiment of the disclosure, the ABP (for example,
either a random asymmetrically branched PEI or a regular
asymmetrically branched PLL) is modified with different kinds of
primary amine groups through, for example, Michael addition or an
addition of acrylic esters onto amines of the polymer. Thus, for
example, through a Michael addition reaction, methyl acrylate, or
other acrylates as provided herein, can be introduced onto the
primary and/or secondary amino groups of, for example, PEI and PLL
homopolymers. The ester groups then can be further derivatized, for
example, by an amidation reaction. Thus, for example, such an
amidation reaction with, for example, ethylenediamine, can yield
the addition of an amino group at the terminus of the newly formed
branch. Other modifications to the polymer can be made using known
chemistries, for example, as provided in "Poly(amines) and
Poly(ammonium salts)" in "Handbook of Polymer Synthesis (Part A),"
Kricheldorf, ed., New York, Marcel Dekker, 1994.
[0098] On such addition, a modified ABP, such as, a modified PEI or
PLL homopolymer, is formed. As an extension of the ABP, such as PEI
and PLL, the resulting modified ABP is also asymmetrically
branched. Depending on the solvent environment (i.e. pH or
polarity), the surface functional groups can carry different charge
and charge density. The molecular shape and functional group
locations (i.e., functional group back folding) then can be further
tuned, based on those characteristic properties.
[0099] In another embodiment, the modified ABP's can be produced
using any of a variety of synthetic schemes that, for example, are
known to be amenable to reaction with a suitable site on the
homopolymer. Moreover, any of a variety of reagents can be used in
a synthetic scheme of choice to yield any of a variety of
modifications or additions to the polymer backbone. Thus, for
example, in the case of the Michael addition reaction to an amine
described above, the addition of any of a variety of substituents
can be used at the alkylation stage, as provided hereinabove, for
example, with an acrylate, which can comprise a saturated or
unsaturated hydrocarbon, such as one comprising one carbon to about
22 carbons, which may be aliphatic, branched, saturated, aromatic,
ringed or combination thereof. Suitable reactants include methyl
acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl
acrylate, hexyl acrylate, heptyl acrylate, octyl acrylate, nonyl
acrylate, decyl acrylate, undecyl acrylate, dodecyl acrylate and
the like, and mixtures thereof. Similarly, at the amidation stage
in the example exemplified above, any of a variety of amines can be
used in the methods provided herein and known in the art. For
example, ethylenediamine, monoethanolamine,
tris(hydroxymethyl)aminomethane, alkyl amine, allyl amine or any
amino modified polymers, including polyethylene glycol (PEG),
perfluoropolymers, polystyrene, polyethylene, polydimethylsilixane,
polyacrylate, polymethylmethacrylate and the like, and mixtures
thereof, can be used. In addition, the linking of the hydrophobic
groups, including aliphatic (e.g., hydrocarbons from C.sub.1 to
about C.sub.22) groups, aromatic groups, polyethylene polymers,
polystyrene polymers, perfluoropolymers, polydimethylsiloxanes,
polyacrylates, polymethylmethacrylates, as well as, hydrophilic
groups, including a OH group, hydrophilic polymers, such as, PEOX,
PEG, PEO etc. to a modified ABP can be achieved by using, for
example, epoxy reactions, amidation reactions, Michael addition
reactions, including using a --SH or an --NH.sub.2 group reacted
with maleimide, aldehyde/ketone-amine/hydrazide coupling reactions,
iodo/iodoacetyl-SH coupling reactions,
hydroxylamine-aldehyde/ketone coupling reactions etc. Such
synthetic strategies allow not only asymmetric growth of the
molecule, where more pockets are introduced, but also the addition
of multiple functional groups at both the interior and the exterior
of the structure. The homopolymer can be modified further using the
same or a different synthetic process until the desired ABP's with
appropriate molecular weight and functional groups are attained. In
addition, the hydrophobic and hydrophilic properties, as well as
charge density of such homopolymers, can be tailored to fit
specific application needs using appropriate monomers for
constructing the homopolymer and suitable modification
reactions.
[0100] In another embodiment of the disclosure, a focal point
(merged from various reactive chain ends during a convergent
synthesis) of a random ABP, such as, polyoxazoline, can be
terminated or reacted with another small molecule to generate
various functional groups at the homopolymeric chain ends,
including primary, secondary or tertiary amines, carboxylate,
hydroxyl, alkyl, fluoroalkyl, aryl, PEG, acetate, amide and/or
ester groups. Alternatively, various initiators also can be
utilized so that the same type of functional group can be
introduced at the surface groups where a polymerization begins
during a convergent synthesis (J. Macromol. Sci. Chem. A9 (5), pp.
703-727 (1975)).
[0101] An alkyl surface modified, randomly branched
poly(2-ethyloxazoline) with a primary amine group at the focal
point of the branched polymer can be prepared using the Litt and
Warakomski procedures, supra. For example,
CH.sub.3(CH.sub.2).sub.17--Br can be utilized as an initiator for
2-ethyloxazoline polymerization through a cationic ring opening
process to generate a randomly branched polymer, followed by
quenching with N-tert-butyloxycarbonylpiperazine (N-Boc-piperazine)
or ethylenediamine (EDA). The termination with a large excess of
EDA allows the hydrophobically modified branched
poly(2-ethyloxazoline) polymer to be functionalized with a primary
amine group at the focal point (FIG. 6B). Alternatively,
N-tert-butyloxycarbonylpiperazine (N-Boc-piperazine) terminated
hydrophobically modified branched poly(2-ethyloxazoline) polymer
also can be deprotected to generate a primary amino group at the
focal point. If not terminated, the focal point of the polymer can
be hydrolyzed to, for example, a hydroxyl group on dissolving in
water (e.g., containing 1N Na.sub.2CO.sub.3).
[0102] While the introduction of a primary amine group to a
hydrophobically modified branched poly(2-oxazoline) homopolymer
enhances drug solubility and produces PAA induced aggregates, the
primary amine group also allows the attachment of various targeting
groups, such as, an antibody, antigen binding portion thereof, an
antigen, or a member of a binding pair to the hydrophobically
modified branched poly(2-oxazoline) polymer (FIG. 10). Such
aggregates or nanoparticles containing such targeting groups and
modifications thereto can provide a targeting ability on the
aggregate with PAA and enables PAA to be released preferentially or
solely at the desired treatment location.
[0103] As taught herein, the MBP's, such as, a hydrophobically
modified homopolymers, including both SBP's and ABP's, can be used
to generate a surface-modified branched polymer for solubilizing
water insoluble or poorly water soluble PAA's, or for forming PAA
induced nanoparticles with water insoluble or poorly water soluble
PAA's, such as, paclitaxel, camptothecin, doxorubin, dolargin,
loperamide, tubocurarine, ibuprofen, diazepam, naproxen,
carbamazepine, griseofulvin, nifedipine, phytosterol, omeprazol,
domperidone, zidovudine, amphotericin B and the like, as well as
drugs described herein, and known to be or are modified to be
poorly soluble in water or insoluble in water. In such a reaction,
the hydrophilic or amphiphilic core can be poly(2-oxazoline),
poly(2-substituted oxazolines), including poly(2-methyloxazoline,
poly(2-ethyloxazoline), poly(2-propyloxazoline) and
poly(2-butyloxazoline) etc., PEG, PEO, polyphosphonate and the
like. The hydrophobic shell can comprise aliphatic hydrocarbons
(such as, from C.sub.1 to about C.sub.22), aromatic hydrocarbons,
polyethylene polymers, polystyrene polymers, perfluoropolymers,
polydimethylsiloxanes, polyacrylates, polymethylmethacrylates and
the like. On the other hand, asymmetrically branched PLL, PEI or
PEOX homopolymers also can be modified with hydrophobic surface
groups listed above to enhance the solubility of water insoluble or
poorly water soluble PAA's.
[0104] The branching density (e.g., from low generation, such as,
star and comb homopolymers, to high generation of dendrimers and
dendrigrafts), as well as the amount of hydrophobic surface group
coverage (e.g., from 0% to 100% coverage) of the branched
homopolymers can affect significantly homopolymer solubility, which
in turn, also affects the ability to dissolve or to adsorb
hydrophobic PAA's. For example, the increase in branching density
and the amount of hydrophobic group coverage will make the
homopolymer more compatible with hydrophobic PAA's.
[0105] In some cases, the ABP's and SBP's with from about 1 to
about 30% or more surface hydrophobic component by weight are
effective at solubilizing or dispersing poorly water soluble or
water insoluble PAA's, such as, paclitaxel. In addition, the
branched homopolymers utilized, for example, a PDX, a PEOX, a PMOX,
PEO/PEG, polyacrylamides, polyphosphates, polyvinylpyrrolidones and
polyvinyl alcohols are soluble in both water and in various organic
solvents, thereby facilitating forming various PAA containing
nanoparticles or aggregates. The good water solubility along with
good hydrophobic drug miscibility in an aqueous solution, with or
without other organic solvents, makes such homopolymers useful for
enhancing the solubility of poorly water soluble PAA's. For
example, the homopolymers of interest simplify manufacturing
processes and decrease production cost by reducing formulation
steps, processing time, as well as the need to use complex and
expensive equipment currently used in the pharmaceutical industry.
If additional branching densities are needed, the SBP's or ABP's
first can be modified with additional groups as described herein,
and then, for example, attached with additional hydrophobic
functional groups for enhancing PAA solubility.
[0106] On mixing hydrophobically modified SBP's or ABP's with a
water insoluble or poorly water soluble PAA, a distinct physical
aggregate is formed of size distinct from aggregates formed only of
polymer (FIGS. 13-15). When the homopolymer and PAA concentrations
decrease, the size and distribution of the polymer PAA aggregates
become much more similar to that of polymer only aggregates
suggesting PAA is released from the induced aggregates or
nanoparticles. The broad size distribution of polymer only
aggregates is similar to that observed for other structures
composed of lipid, whether or not associated with a PAA. On the
other hand, the PAA induced aggregates of interest are of a
particular size of narrower distribution, that is, unique
aggregates of certain size are produced. As PAA concentration in
the aggregate decreases, homopolymer concentration in the aggregate
decreases, aggregate concentration decreases or any combination
thereof, the aggregates of interest release PAA, as evidenced by a
reduction of aggregate size and/or a broader distribution of
aggregate size. The broader distribution may result from a mixture
of homopolymer only aggregates and polymer PAA aggregates of
varying size due to PAA release, until the only aggregates observed
are those which have the characteristics of those which are
homopolymer only. In other words, the PAA is released gradually
after introduced into a host, such as, in the circulatory system.
That mechanism is important for various drug delivery applications
including, intravenous (IV), oral, transdermal, ocular,
intramuscular and the like modes of administration, and where a
delayed release or sustained release profile may be desirable.
[0107] The PAA induced aggregates also can be linked with a
targeting moiety or group including, but not limited to, an
antibody (or antigen-binding portion thereof), antigen, cognate
carbohydrates (e.g., sialic acid), a cell surface receptor ligand,
a moiety that binds a cell surface receptor, a moiety that binds a
cell surface saccharide, an extracellular matrix ligand, a
cytosolic receptor ligand, a growth factor, a cytokine, an
incretin, a hormone, a lectin, a lectin target, such as, a
galactose, a galactose derivative, an N-acetylgalactosamine, a
mannose, a mannose derivative and the like, a vitamin, such as, a
folate, a biotin and the like, an avidin, a streptavidin, a
neutravidin, a DNA, an RNA etc. to form a conjugate so that the
targeting group(s) are incorporated with nanocomposite particle of
interest (FIG. 10).
[0108] In addition, a diagnostic agent, such as, an imaging agent,
a radionuclide or any of a variety of contrasting agents also can
be carried by said aggregates of interest. Thus, a combination of
chemotherapy, radiotherapy and/or targeted therapy with real time
diagnostic/monitoring capabilities can be achieved (FIGS. 11 and
12). In some embodiments, the diagnostic agent is poorly soluble or
water insoluble, thereby negating the need, for example, of a PAA
of interest to induce aggregation.
[0109] Thus, a diagnostic agent can be a metal containing material
or a paramagnetic material, e.g., magnetic resonance imaging
materials, which can be deposited on the surface or entrapped
within a nanocomposite of interest so that the nanoparticle can be
used as both a diagnostic and a therapeutic agent. In yet another
aspect of the disclosure, the imaging material containing
nanoparticles further can comprise a targeting moiety/group, which
allows such nanoparticle to target specific locations that need
therapeutic treatment, for example, a tumor site.
[0110] Thus, a molecule with the ability to bind another molecule,
such as a biological polymer, such as a polypeptide, or a
polysaccharide, an enzyme, a receptor and the like, which can bind
a vitamin, a lectin, a metal and so on, can be used in a composite
of interest. Metals and metal ions that can be carried by a polymer
of interest may include, but are not limited to, transition metals,
such as Sc, Y, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Tc, Re, Fe,
Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd Hg, Ga, In or
Tl, alkali metals, alkaline earth metals, Lanthanide series
elements, such as Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm,
Yb and Lu, Actinide series elements, such as Th, Pa, U, Np, Pu, Am,
Cm, Bk, Cf, Es, Fm, Md, No and Lr, and the like.
[0111] Such can be carried by, for example, one or more chelating
groups, including, but not limited to, ethylenediaminetetraacetic
acid (EDTA), diethylenetriaminepentaacetic acid (DTPA),
diethylenetriaminepentaacetic acid (DTPA),
1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA),
1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A),
1-oxa-4,7,10-triazacyclododecane-triacetic acid (DOXA),
1,4,7-triazacyclononanetriacetic acid (NOTA),
1,4,8,11-tetraazacyclotetradecanetetraacetic acid (TETA),
DOTA-N(2-aminoethyl)amide and DOTA-N-(2-aminophenethyl)amide.
[0112] For such diagnostic purposes, a conjugate of interest
comprises a reporter molecule that can be detected by an external
device, such as, a gamma camera. Thus, a conjugate can be
configured to comprise, for example, a radioisotope that will emit
detectable radiation. The conjugate is placed into a format
suitable for consumption or placement in a body, employing reagents
suitable therefor as known in the art. The conjugate composition is
administered as known in the art, such as orally, rectally,
intravenously and so on.
[0113] In another aspect of the disclosure, the nanoparticle can
carry different types of PAA's so that a combination or cocktail
therapy can be achieved. Such PAA's may include, but are not
limited to, various small molecule drugs, inorganic drugs and
biological molecule based drugs, such as, a peptide, a protein, an
antibody, an enzyme, a vaccine and the like. The second or more
PAA's need not be poorly soluble or water insoluble as the second
or more PAA's can be situated within voids in the aggregate and
need not be located at the surface. Hence, any of the PAA's noted
herein or known in the art can comprise the second or more
PAA's.
Drug Formulation and Nanoparticle Preparation
[0114] PAA and modified homopolymer can be suspended individually
in suitable buffers and/or solvents, such as, a buffer, acetone,
ethanol and the like, at suitable concentrations, such as those
which are established for in vivo use, generally in milligram or
nanogram quantities. Then, the two solutions are mixed at a
suitable temperature, such as, room temperature or at another
temperature known to be acceptable for maintaining integrity of the
PAA and homopolymer, for a suitable period of time, such as, one
hour, two hours and so on. Other incubation times can vary from
minutes to hours as the aggregates of interest are stable once
formed. The aggregates can be concentrated or collected practicing
methods known in the art, for example, by filtration,
centrifugation, evaporation, lyophilization, dialysis and the like.
The aggregates can be desiccated for extended shelf life.
[0115] For example, paclitaxel was dissolved in ethanol in various
amounts of up to 40 mg/mL. A hydrocarbon
(CH.sub.3(CH.sub.2).sub.17) modified randomly branched PEOX was
prepared as taught herein and dissolved at varying concentrations
of up 100 mg/mL in saline.
[0116] The two solutions then were mixed in various volumes to
result in final homopolymer to paclitaxel molar ratios in the
mixtures ranging from 3:1 to 10:1. The mixtures subsequently were
frozen at -80.degree. C. for 3 hours then lyophilized for 20 to 48
hours depending on volume to yield a dry powder.
[0117] The size of the aggregates or nanoparticles, as measured by
light scattering, can range from about 120 nm (e.g., at 3 mg
paclitaxel per mL) to about 165 nm (e.g., at 5 mg paclitaxel per
mL) in diameter depending on the concentration of drug and
concentration of homopolymer.
[0118] Alternatively, PAA and homopolymer can be dissolved in a
common solvent, which generally is not necessarily hydrophilic but
is miscible with water, and then added to an aqueous solution.
Hence, paclitaxel and PEOX can be dissolved in acetone and then
dropwise added to water under agitation, such as, while stirred or
sonicated, followed by dialysis with a 1000 MW cutoff membrane. The
final product then can be lyophilized.
[0119] A conjugate of interest can be incorporated into
pharmaceutical compositions suitable for administration, for
example, for diagnostic imaging, for lifestyle management or to
attain a therapeutic milestone. Such compositions typically
comprise an aggregate of interest and a pharmaceutically acceptable
carrier, excipient or diluent, which is intended to include any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents and the
like, that is, those ingredients of a pharmaceutically acceptable
composition aside from the PAA's that are included therein for
particular purposes, such as, bulking, preservation, delayed
release, binding and so on, as known in the art, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agents are incompatible with
the active compound, use thereof in the compositions is
contemplated. Supplementary active compounds also can be
incorporated into the composition.
[0120] A pharmaceutical composition of the disclosure for use as
disclosed herein is formulated to be compatible with the intended
route of administration. Examples of routes of administration
include parenteral, e.g., intravenous, intradermal, subcutaneous,
oral (e.g., inhalation), transdermal (topical), transmucosal and
rectal administration. Solutions or suspensions used for
parenteral, intradermal or subcutaneous application can include a
sterile diluent, such as, water for injection, saline, oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents, such as, benzyl alcohol
or methyl parabens; antioxidants, such as, ascorbic acid or sodium
bisulfate; chelating agents, such as, EDTA; buffers, such as,
acetates, citrates or phosphates; and agents for the adjustment of
tonicity, such as, sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as HCl or NaOH. The parenteral
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic as an article of
manufacture. Generally, an in vivo diagnostic agent will be
administered orally, rectally, intravenously, intraperitoneally and
so on.
[0121] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water or
phosphate-buffered saline (PBS). The composition generally is
sterile and is fluid to the extent that easy syringability exists.
The composition must be stable under the conditions of manufacture
and storage and must be preserved against the contaminating action
of microorganisms, such as, bacteria and fungi. The carrier can be
a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, liquid
polyethylene glycol and the like) and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal
and the like. Isotonic agents, for example, sugars, polyalcohols,
such as, mannitol, sorbitol or sodium chloride can be included in
the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent that delays absorption, for example, aluminum monostearate
or gelatin.
[0122] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount of an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound in a sterile vehicle that contains a basic dispersion
medium and the required other ingredients, for example, from those
enumerated above, and as known in the art. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preparation can be prepared by, for example, lyophilization, vacuum
drying or freeze drying, that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The preparation of interest can
be stored and reconstituted with a suitable liquid for use.
[0123] Oral compositions generally include an inert diluent,
flavorant, odorant or an edible carrier. The composition can be
enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral therapeutic administration, the active compound can
be incorporated with excipients and used in the form of tablets,
troches or capsules. Oral compositions also can be prepared using a
fluid carrier to yield a syrup or liquid formulation, or for use as
a mouthwash, wherein the compound in the fluid carrier is applied
orally and swished and expectorated or swallowed.
[0124] Pharmaceutically compatible binding agents and/or adjuvant
materials can be included as part of the composition. Tablets,
pills, capsules, troches and the like can contain a binder, such
as, microcrystalline cellulose, gum tragacanth or gelatin; an
excipient, such as, starch or lactose, a disintegrating agent, such
as, alginic acid, Primogel or corn starch; a lubricant, such as,
magnesium stearate or Sterotes; a glidant, such as, colloidal
silicon dioxide; a sweetening agent, such as, sucrose or saccharin;
or a flavoring agent, such as, peppermint, methyl salicylate or
orange flavoring.
[0125] For administration by inhalation, the compound is delivered
in the form of, for example, a wet or dry aerosol spray from a
pressurized container or dispenser that contains a suitable
propellant, e.g., a gas, such as, carbon dioxide or a nebulizer, or
a mist.
[0126] Systemic administration also can be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants generally are known in the art and
include, for example, for transmucosal administration, detergents,
bile salts and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels or creams as
generally known in the art. A suitable carrier includes
dimethylsulfoxide.
[0127] The compound also can be prepared in the form of
suppositories (e.g., with conventional suppository bases, such as,
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0128] In one embodiment, the active compound is prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters and
polylactic acid.
[0129] Methods for preparation of such formulations will be
apparent to those skilled in the art. The materials also can be
obtained commercially, for example, from Alza Corporation and Nova
Pharmaceuticals, Inc.
[0130] The instant aggregates can be used in topical forms, such
as, creams, ointments, lotions, unguents, other cosmetics and the
like. PAA's and other bioactive or inert compounds can be carried,
and include emollients, bleaching agents, antiperspirants,
pharmaceuticals, moisturizers, scents, colorants, pigments, dyes,
antioxidants, oils, fatty acids, lipids, inorganic salts, organic
molecules, opacifiers, vitamins, pharmaceuticals, keratolytic
agents, UV blocking agents, tanning accelerators, depigmenting
agents, deodorants, perfumes, insect repellants and the like.
[0131] It can be advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for a subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce a desired therapeutic
endpoint.
[0132] The dosages, for example, preferred route of administration
and amounts are obtainable based on empirical data obtained from
preclinical and clinical studies, practicing methods known in the
art. The dosage and delivery form can be dictated by and can be
dependent on the characteristics of the PAA, the polymer, the
particular therapeutic effect to be achieved, the characteristics
and condition of the recipient and so on. For repeated
administrations over several days or longer, depending on the
condition, the treatment can be sustained until a desired endpoint
is attained. An exemplary dosing regimen is disclosed in WO
94/04188.
[0133] The progress of the therapy can be monitored by conventional
techniques and assays, as well as patient input.
[0134] The pharmaceutical compositions can be included in a
container, pack or dispenser together with instructions for
administration.
[0135] Another method of administration comprises the addition of a
compound of interest into or with a food or drink, as a food
supplement or additive, or as a dosage form taken on a prophylactic
basis, similar to a vitamin. The aggregate of interest can be
encapsulated into forms that will survive passage through the
gastric environment. Such forms are commonly known, for example,
enteric coated formulations. Alternatively, the aggregate of
interest can be modified to enhance half life, such as, chemical
modification or combination with agents known to result in delayed,
sustained or controlled release, as known in the art.
[0136] The instant disclosure now will be exemplified in the
following non-limiting examples.
EXAMPLES
Materials
[0137] Symmetrically branched PPI dendrimers were purchased from
Sigma-Aldrich. Symmetrically branched PEI dendrimers and
dendrigrafts were prepared according to procedures provided in U.S.
Pat. Nos. 4,631,337, 5,773,527, 5,631,329 and 5,919,442. All of the
antibodies were purchased from Sigma-Aldrich, Biodesign or
Fitzgerald. Different generation PAMAM dendrimers were purchased
from Dendritech, Inc.
Synthesis of Modified Symmetrically Branched PPIs with Amino
Functional Groups (m-SB-PPI-NH.sub.2-1.0)
[0138] The following reagents including symmetrically branched PPI
(SB-PPI-4, 8, 16, 32, 64, MW 316, 773, 1,687, 3,514 and 7,168),
methyl acrylate (MA, FW=86.09), ethylenediamine (EDA, FW=60.10) and
methanol were utilized.
[0139] To a round bottom flask were added 1.0 g PPI-64 dendrimer
(MW 7168) and 20 ml methanol (solution A). To a separate round
bottom flask were added 2.4 g methylacrylate (MA) and 10 ml
methanol (solution B). Solution A was then slowly dropped into
solution B while stirring at room temperature. The resulting
solution was allowed to react at 40.degree. C. for 2 hours. On
completion of the reaction, the solvent and unreacted MA monomer
were removed by rotary evaporation and the product, 2.5 g of MA
functionalized PPI, then was redissolved in 20 ml of methanol.
[0140] To a round bottom flask were added 160 g EDA and 50 ml of
methanol, followed by a slow addition of MA functionalized PPI at
0.degree. C. The solution then was allowed to react at 4.degree. C.
for 48 hours. The solvent and the excess EDA were removed by rotary
evaporation. The crude product then was precipitated from an ethyl
ether solution and further purified by dialysis to give about 2.8 g
of primary amine-functionalized symmetrically branched PPI
(m-SB-PPI-NH.sub.2-1.0) with a molecular weight of about 21,760.
The product was characterized by .sup.1H and .sup.13C nuclear
magnetic resonance (NMR) and size exclusion chromatography
(SEC).
[0141] Other MA or primary amine-modified symmetrically branched
PPI dendrimers and symmetrically branched PEI dendrigrafts with
various molecular weights were prepared in a similar manner.
Synthesis of Modified Symmetrically Branched PPIs with Mixed
Hydroxyl and Amino Functional Groups
(mix-m-SB-PPI-64-NH.sub.2/OH-2)
[0142] Amino functionalized symmetrically branched PPI
(m-SB-PPI-64-NH.sub.2-1.0), MA, EDA, monoethanolamine (MEA,
FW=61.08) and methanol were utilized.
[0143] To a round bottom flask were added 1.0 g amino-modified PPI
or m-SB-PPI-NH.sub.2-1.0 produced from the previous procedure and
20 ml of methanol (solution A). To a separate round bottom flask
were added 2.4 g of MA and 10 ml methanol (solution B). Solution A
was then slowly dripped into solution B while stirring at room
temperature. The resulting solution was allowed to react at
40.degree. C. for 2 hours. On completion of the reaction, the
solvent and unreacted monomer MA were removed by rotary evaporation
and the product, 2.5 g of MA functionalized m-SB-PPI-64-MA-1.5,
then was redissolved in 20 ml of methanol.
[0144] To a round bottom flask were added 32 g EDA, 130 g MEA and
100 ml methanol (the mole ratio of EDA:MEA was 20:80), followed by
slow addition of m-SB-PPI-64-MA-1.5 at 0.degree. C. The solution
then was allowed to react at 4.degree. C. for 48 hours. The solvent
and the excess EDA were removed by rotary evaporation. The crude
product then was precipitated from an ethyl ether solution and
further purified by dialysis to give about 2.8 g of mixed hydroxyl
and amino functionalized (mixed surface) SBP
(mix-m-SB-PPI-64-NH.sub.2/OH-2.0, with an average of 20% NH.sub.2
and 80% OH surface groups and a molecular weight of about
21,862).
[0145] Other modified random AB-PEI and regular AB PLL molecules
with varying ratios of hydroxyl and amino groups, as well as
different molecular weights, were prepared in a similar manner.
[0146] Random asymmetrically branched PEI's were purchased from
Aldrich and Polysciences. Regular ABP's were prepared according to
procedures provided in U.S. Pat. No. 4,289,872. All of the
antibodies were purchased from Sigma-Aldrich, Biodesign or
Fitzgerald.
Synthesis of Modified Random Asymmetrically Branched PEIs with
Amino Functional Groups (m-ran-AB-PEI-NH.sub.2-1.0)
[0147] Random asymmetrically branched PEI (ran-AB-PEI, MW 2,000,
25,000 and 75,000), MA, EDA and methanol were utilized.
[0148] To a round bottom flask were added 1.0 g PEI (MW 2,000) and
20 ml methanol (solution A). To a separate round bottom flask were
added 3.0 g MA and 10 ml methanol (solution B). Solution A was then
slowly dripped into solution B while stirring at room temperature.
The resulting solution was allowed to react at 40.degree. C. for 2
hours. On completion of the reaction, the solvent and unreacted MA
were removed by rotary evaporation and the product, MA
functionalized PEI, then was redissolved in 20 ml of methanol.
[0149] To a round bottom flask were added 80 g EDA and 50 ml of
methanol, followed by a slow addition of MA-functionalized PEI at
0.degree. C. (1 g MA dissolved in 20 ml methanol). The solution
then was allowed to react at 4.degree. C. for 48 hours. The solvent
and excess EDA were removed by rotary evaporation. The crude
product then was precipitated from an ethyl ether solution and
further purified by dialysis to give about 3.0 g of primary
amine-functionalized random asymmetrically branched PEI
(m-ran-AB-PEI-NH.sub.2-1.0) with a molecular weight of about 7,300.
The product was characterized by .sup.1H and .sup.13C NMR and
SEC.
[0150] Other MA or primary amine modified random asymmetrically
branched PEI and regular asymmetrically branched PLL polymers with
various molecular weights were prepared in a similar manner.
Modification of Branched Polymers with Hydrocarbon Chains
[0151] The modification of a randomly branched PEI with 10%
hydrocarbon chains is used as an example. One gram of branched PEI
(FW=25000) was dissolved in 10 mL methanol. To the solution were
added 0.23 g of 1,2-epoxyhexane (FW=100.16) and the mixture was
heated at 40.degree. C. for 2 hours. The solvent then was rotary
evaporated and the residue redissolved in water. After dialysis
(3,500 cutoff), the modified PEI was generated. Other MBP's, such
as, PAMAM, PEI and PPI dendrimers and dendrigrafts, and asymmetric
PLL with various percentages and lengths (e.g., C.sub.4, C.sub.12,
C.sub.18 and C.sub.22) of hydrocarbon chains were prepared in a
similar manner.
Synthesis of Modified Random Asymmetrically Branched PEIs with
Mixed Hydroxyl and Amino Functional Groups
(m-ran-AB-PEI-NH.sub.2/OH-2)
[0152] Amino functionalized random asymmetrically branched PEI
(m-ran-AB-PEI-NH.sub.2-1.0), MA, EDA, monoethanolamine (MEA,
FW=61.08) and methanol were utilized.
[0153] To a round bottom flask were added 1.0 g amino-modified PEI
or m-ran-AB-PEI-NH.sub.2-1.0 produced from the previous procedure
and 20 ml of methanol (solution A). To a separate round bottom
flask were added 3.0 g of MA and 10 ml methanol (solution B).
Solution A then was slowly dripped into solution B while stirring
at room temperature. The resulting solution was allowed to react at
40.degree. C. for 2 hours. On completion of the reaction, the
solvent and unreacted MA were removed by rotary evaporation and the
product, MA functionalized m-ran-AB-PEI-MA-1.5, then was
redissolved in 20 ml of methanol.
[0154] To a round bottom flask were added 60 g EDA, 244 g MEA and
100 ml methanol (the mole ratio of EDA:MEA was 20:80), followed by
slow addition of m-ran-AB-PEI-MA-1.5 at 0.degree. C. (1 g MA
dissolved in 20 ml of methanol). The solution then was allowed to
react at 4.degree. C. for 48 hours. The solvent and excess EDA were
removed by rotary evaporation. The crude product then was
precipitated from an ethyl ether solution and further purified by
dialysis to give about 2.4 g of mixed hydroxyl and amino
functionalized random ABP (m-ran-AB-PEI-NH.sub.2/OH-2.0, with an
average of 20% NH.sub.2 and 80% OH surface groups and the molecular
weight was about 18,000).
[0155] Other modified random AB-PEI and regular AB polylysine
polymers with various ratios of hydroxyl and amino groups, as well
as different molecular weights were prepared in a similar
manner.
Synthesis of Alkyl-Modified Random Asymmetrically Branched
Poly(2-ethyloxazoline) (PEOX) with Primary Amine Chain End
Group
[0156] The synthesis of CH.sub.3--(CH.sub.2).sub.11-PEOX-ABP100
(ABP100 is an arbitrary name to denote the ratio of monomer to
initiator in the initial reaction) is provided as a general
procedure for the preparation of core shell structures. A mixture
of CH.sub.3(CH.sub.2).sub.11--Br (2.52 g) in 500 ml of toluene was
azeotroped to remove water with a distillation head under N.sub.2
for about 15 min. 2-Ethyloxazoline (100 g) was added dropwise
through an addition funnel and the mixture was allowed to reflux
between 24 and 48 hours. On completion of the polymerization, 12.12
g of EDA were added to the reactive polymer solution (A) to
introduce the amine function group. The molar ratio of
polyoxazoline chain end to EDA was 1 to 20.
[0157] N-tert-butyloxycarbonylpiperazine (N-Boc-piperazine) or
water (e.g., with 1N Na.sub.2CO.sub.3) can be added to terminate
the reaction. Morpholine or PEI also can be added to the reactive
polymer solution (A) to terminate the reaction. The crude product
was redissolved in methanol and then precipitated from a large
excess of diethyl ether. The bottom layer was redissolved in
methanol and dried by rotary evaporation and vacuum to give an
asymmetrically random branched PEOX polymer or PEOX-PEI copolymer
as a white solid (101 g). Other asymmetrically randomly branched
polymers, such as, C.sub.6-PEOX ABP20, 50, 100, 200, 300, 500,
C.sub.18-PEOX ABP20, 50, 200, 300, 500, C.sub.22-PEOX ABP20, 50,
100, 200, 300, 500, and polystyrene-PEOX etc., as well as,
non-modified and modified poly(2-substituted oxazoline), such as,
poly(2-methyloxazoline), were prepared in a similar manner. All the
products were analyzed by SEC and NMR.
Preparation of Mixed Surface Modified Symmetrical Branched
Polymer-IgG Conjugates
[0158] The preparation of mixed surface (OH/NH.sub.2 mix) modified
symmetrically branched PPI-IgG conjugates
(mix-m-SB-PPI-64-NH.sub.2/OH-2-IgG conjugates) is provided as a
general procedure for the preparation of polymer antibody and
polymer streptavidin conjugates. Other conjugates, such as,
m-SB-PPI-4-NH.sub.2-1-IgG, m-SB-PPI-8-NH.sub.2-1-IgG,
m-SB-PPI-16-NH.sub.2-1-IgG, m-SB-PPI-32-NH.sub.2-1-IgG,
m-SB-PPI-4-NH.sub.2-2-IgG, m-SB-PPI-8-NH.sub.2-2-IgG,
m-SB-PPI-16-NH.sub.2-2-IgG, m-SB-PPI-32-NH.sub.2-2-IgG,
m-SB-PPI-4-NH.sub.2-3-IgG, m-SB-PPI-8-NH.sub.2-3-IgG,
m-SB-PPI-16-NH.sub.2-3-IgG, m-SB-PPI-32-NH.sub.2-3-IgG,
mix-m-SB-PPI-4-NH.sub.2/OH-1 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-8-NH.sub.2/OH-1 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-16-NH.sub.2/OH-1 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-32-NH.sub.2/OH-1 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-4-NH.sub.2/OH-2 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-8-NH.sub.2/OH-2 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-16-NH.sub.2/OH-2 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-32-NH.sub.2/OH-2 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-4-NH.sub.2/OH-3 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-8-NH.sub.2/OH-3 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-16-NH.sub.2/OH-3 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-32-NH.sub.2/OH-3 (OH/NH.sub.2 mix)-IgG, as well as
primary amine and mix OH/NH.sub.2 modified combburst PEI
dendrigrafts (Generation 0-5) also were obtained in a similar
manner. The synthesis of other protein attached to a modified SBP
of interest also was obtained in a similar manner. The
biotinylated-IgG conjugates were synthesized as provided in
Bioconjugate Techniques (G. Hermanson, Academic Press, 1996).
LC-SPDP-Mixed Surface m-SB-PPI-64-NH.sub.2/OH-2
[0159] To the mixed surface randomly branched
mix-m-SB-PPI-64-NH.sub.2/OH-2 (4.times.10.sup.-7 mol) in 400 .mu.l
of phosphate buffer (20 mM phosphate and 0.1 M NaCl, pH 7.5) were
added 4.0.times.10.sup.-6 mol of sulfo-LC-SPDP (Pierce, Ill.) in
400 .mu.L of water. The mixture was vortexed and incubated at
30.degree. C. for 30 minutes. The
LC-SPDP-mix-m-SB-PPI-64-NH.sub.2/OH-2 was purified by gel
filtration chromatography and equilibrated with buffer A (0.1 M
phosphate, 0.1 M NaCl and 5 mM EDTA, pH 6.8). The product was
concentrated further to yield 465 .mu.L of solution with a
concentration of approximately 0.77 nmol.
Thiolated Mix m-SB-PPI-64-NH.sub.2/OH-2 from LC-SPDP
Mix-m-SB-PPI-64-NH.sub.2/OH-2
[0160] The LC-SPDP mix-m-SB-PPI-64-NH.sub.2/OH-2 (50 nmol in 65
.mu.l of buffer A) was mixed with 100 .mu.L of dithiothreitol (DTT)
(50 mM in buffer A) and was incubated at room temperature for 15
minutes. Excess DTT and byproducts were removed by gel filtration
with buffer A. The product was concentrated in a 10 K Centricon
Concentrator to yield 390 .mu.L of the thiolated
mix-m-SB-PPI-64-NH.sub.2/OH-2 that was used for conjugation with
activated antibody.
Maleimide R (MAL-R)-Activated Antibody
[0161] To the antibody in PBS (310 .mu.L, 5.1 mg or 34 nmol) were
added 20.4 .mu.L of a MAL-R-NHS (N-hydroxysuccinimide) solution (10
mM in water). The mixture was vortexed and incubated at 30.degree.
C. for 15 minutes. The product was purified by gel filtration with
buffer A. The maleimide-R-activated antibody was used for
conjugation with the thiolated mix-m-SB-PPI-64-NH.sub.2/OH-2.
Mix-m-SB-PPI-64-NH.sub.2/OH-2-Antibody Conjugate
[0162] To the thiolated mix-m-SB-PPI-64-NH.sub.2/OH-2 (310 .mu.L or
35.7 nmol) was added the MAL-R-activated antibody (4.8 mL or 34
nmol). The reaction mixture was concentrated to approximately 800
.mu.L and then allowed to incubate overnight at 4.degree. C. and/or
at room temperature for about 1 hr. On completion, the reaction was
quenched with 100 .mu.L of ethyl maleimide (50 mmolar solution) and
the conjugate then was fractionated on a carboxymethyl cellulose
column (5 mL) with a sodium chloride step gradient in 20 mM
phosphate buffer at pH 6. The conjugate was eluted with a sodium
chloride gradient and characterized by cationic exchange
chromatography, UV spectroscopy and polyacrylamide gel
electrophoresis.
Conjugation Via Reductive Coupling
Reduction of Antibody
[0163] To the antibody, 2.1 mg or 14 nmol in 160 .mu.L of buffer B
(containing 0.1 M sodium phosphate, 5 mM EDTA and 0.1 M NaCl, pH
6.0) were added 40 .mu.L of DTT (50 mM in buffer B). The solution
was allowed to stand at room temperature for 30 min. The product
was purified by gel filtration in a Sephadex G-25 column
equilibrated with buffer B. The reduced antibody was concentrated
to 220 .mu.l, and was used for conjugation.
MAL-R-Mixed Surface Modified SBP
[0164] To the mixed surface modified SBP in 400 .mu.L
(400.times.10.sup.-9 mols) at pH 7.4 were added 400 .mu.L of
MAL-R-NHS (10 mM in water). That was mixed and incubated at
30.degree. C. for 15 min. On termination, the product was purified
on a Sephadex G-25 column equilibrated with buffer B. The
MAL-R-mixed surface modified SBP was collected and stored in
aliquots in the same buffer at -40.degree. C.
Mixed Surface Modified SBP-Antibody Conjugate
[0165] To the reduced antibody (14 nmols in 220 .mu.l) was added
the MAL-R-mix-m-SB-PPI-64-NH.sub.2/OH-2 (154 .mu.L, 16.6 nmols)
with stirring. The pH was adjusted to about 6.8 by the addition of
12.5 .mu.L of sodium carbonate (1.0 M solution), the reaction was
continued for 1 hr at room temperature and terminated with the
addition of 100 .mu.L of cysteamine (0.4 mM solution). The
conjugation mixture was purified on a CM cellulose column with a
sodium chloride gradient elution.
Preparation of IgG-Asymmetrical Randomly Branched Polymer
Conjugates
[0166] The preparation of randomly branched mixed surface
(OH/NH.sub.2 mix) m-ran-AB-PEI-NH.sub.2/OH-2-IgG conjugates is
provided as a general procedure for the preparation of
polymer-antibody and polymer-streptavidin conjugates. Other
conjugates such as PEI-IgG, m-ran-AB-PEI-NH.sub.2-1-IgG,
m-ran-AB-PEI-NH.sub.2-2-IgG, m-ran-AB-PEI-NH.sub.2-3-IgG,
m-ran-AB-PEI-NH.sub.2-4-IgG, as well as m-ran-AB-PEI-NH.sub.2/OH-1
(OH/NH.sub.2 mix)-IgG, m-ran-AB-PEI-NH.sub.2/OH-2 (OH/NH.sub.2
mix)-IgG, m-ran-AB-PEI-NH.sub.2/OH-3 (OH/NH.sub.2 mix)-IgG, regular
polylysine polymer, alkyl modified random branched
poly(2-ethyloxazoline) with primary amine chain ends were all
synthesized in a similar manner. The synthesis of various protein
conjugates with asymmetrically random branched PEOX polymers also
is conducted in a similar manner. The biotinylated-IgG conjugates
were synthesized as provided in Bioconjugate Techniques (G.
Hermanson, Academic Press, 1996).
LC-SPDP-Mixed Surface m-ran-AB-PEI-NH.sub.2/OH-2
[0167] To the mixed surface randomly branched
m-ran-AB-PEI-NH.sub.2/OH-2 (4.times.10.sup.-7 mol) in 400 .mu.L of
phosphate buffer (20 mM phosphate and 0.1 M NaCl, pH 7.5) were
added 4.0.times.10.sup.-6 mol of sulfo-LC-SPDP (Pierce, Ill.) in
400 .mu.l of water. That was vortexed and incubated at 30.degree.
C. for 30 minutes. The LC-SPDP-m-ran-AB-PEI-NH.sub.2/OH-2 was
purified by gel filtration chromatography and equilibrated with
buffer A (0.1 M phosphate, 0.1 M NaCl and 5 mM EDTA, pH 6.8). The
product was concentrated further to yield 465 .mu.l of solution
with a concentration of approximately 0.77 nmol/.mu.mol.
Thiolated m-ran-AB-PEI-NH.sub.2/OH-2 from LC-SPDP
m-ran-AB-PEI-NH.sub.2/OH-2
[0168] The LC-SPDP m-ran-AB-PEI-NH.sub.2/OH-2 (50 nmol in 65 ml of
buffer A) was mixed with 100 .mu.L of dithiothreitol (DTT) (50 mM
in buffer A) and was allowed to incubate at room temperature for 15
minutes. Excess DTT and byproducts were removed by gel filtration
with buffer A. The product was concentrated in a 10 K Centricon
Concentrator to yield 390 .mu.L of the thiolated
m-ran-AB-PEI-NH.sub.2/OH-2 that was used for conjugation with
activated antibody.
[0169] Maleimide-R-activated antibody made as described above was
used for conjugation with the thiolated
m-ran-AB-PEI-NH.sub.2/OH-2.
m-ran-AB-PEI-NH.sub.2/OH-2-Antibody Conjugate
[0170] To the thiolated m-ran-AB-PEI-NH.sub.2/OH-2 (310 .mu.L or
35.7 nmol) was added the MAL-R-activated antibody (4.8 mL or 34
nmol). The reaction mixture was concentrated to approximately 800
.mu.L and allowed to incubate overnight at 4.degree. C. and/or at
room temperature for about 1 hr. On completion, the reaction was
quenched with 100 .mu.L of ethyl maleimide (50 mmolar solution) and
the conjugate then was fractionated on a carboxymethyl cellulose
column (5 ml) with a sodium chloride step gradient in 20 mM
phosphate buffer at pH 6. The conjugate was eluted with a sodium
chloride gradient and characterized by cationic exchange
chromatography, UV spectroscopy and polyacrylamide gel
electrophoresis.
Paclitaxel Formulation and Nanoparticle Preparation
[0171] Paclitaxel was dissolved in ethanol to a concentration of up
to 40 mg/mL.
[0172] A C.sub.18 hydrocarbon modified randomly branched PEOX was
prepared as taught herein.
[0173] The polymer was separately dissolved to a concentration of
up to 100 mg/mL in saline. The two solutions were then mixed at
various volumes to result in final polymer to paclitaxel molar
ratios in the mixtures ranging from 3:1 to 10:1. The mixtures were
subsequently frozen at -80.degree. C. for 3 hours then lyophilized
for 20 to 48 hours depending on volume.
[0174] The size of the aggregates as measured by light scattering
ranged from about 120 nm to about 165 nm in diameter.
[0175] Alternatively, both paclitaxel and the PEOX polymer can be
dissolved in a common solvent, such as, acetone and then dropwise
added to water while being stirred or sonicated, followed by
dialysis with a 1000 MW cutoff membrane. The final product then can
be generated by lyophilization and the size of the aggregates was
measured by light scattering.
[0176] Other PAA induced aggregates or nanoparticles using various
hydrophobically surface modified branched polymers, such as,
C.sub.4, C.sub.6, C.sub.12 or C.sub.22 hydrocarbon modified
randomly branched PEOX, PEI and PPI polymers; C.sub.4, C.sub.6,
C.sub.12, C.sub.18 and C.sub.22 hydrocarbon modified PAMAM, PEI and
PPI dendrimers and dendrigrafts; and C.sub.4, C.sub.6, C.sub.12,
C.sub.18 and C.sub.22 hydrocarbon modified branched PLL/polymers
can be prepared in a similar manner.
[0177] Thus, C.sub.18-PEOx-100-NH.sub.2 (500 mg) is dissolved in 5
mL of ethanol to yield a 100 mg/mL solution. A 20 mg/mL solution of
Paclitaxel is also prepared by dissolving 100 mg in 5 mL of
ethanol. The two solutions are mixed for 20 minutes resulting in a
solution containing 10 mg Paclitaxel and 50 mg polymer per mL,
providing a solution with a 1:5 drug:polymer ratio. The mixture is
placed on a rotary evaporator and the ethanol removed to dryness.
The resultant solid is redissolved with stirring in 33 mL of saline
solution to a final Paclitaxel concentration of 3 mg/mL. The
solution preparation is passed through a 0.8 .mu.m filter and then
a 0.22 .mu.m filter. The filtrate is frozen in a vial at
-70.degree. C. for at least 2 hours then lyophilized over a 72 hour
period. The vial is stoppered and the ready-to-use white powder is
stored at room temperature.
Nanoparticle Measurement
[0178] The size of various polymers, polymer only aggregates, as
well as drug-induced polymer aggregates was measured by a dynamic
light scattering method using a Malvern Zetasizer Nano-ZS Zen3600
particle size analyzer.
Activity Testing
[0179] Metabolism in viable cells produces "reducing equivalents,"
such as, NADH or NADPH. Such reducing compounds pass electrons to
an intermediate electron transfer reagent that can reduce the
tetrazolium product, MTS (Promega), into an aqueous, soluble
formazan product, which is colored. At death, cells rapidly lose
the ability to reduce tetrazolium products. The production of the
colored formazan product, therefore, is proportional to the number
of viable cells in culture.
[0180] The CellTiter 96.RTM. Aqueous products (Promega) are MTS
assays for determining the number of viable cells in culture. The
MTS tetrazolium is similar to MTT tetrazolium, with the advantage
that the formazan product of MTS reduction is soluble in cell
culture medium and does not require use of a solubilization
solution. A single reagent added directly to the assay wells at a
recommended ratio of 20 .mu.l reagent to 100 .mu.l of culture
medium was used. Cells were incubated 1-4 hours at 37.degree. C.
and then absorbance was measured at 490 nm.
[0181] Thus, the cytotoxicity of various paclitaxel containing
aggregates of interest, along with commercially available Taxol and
Abraxane, a paclitaxel nanoparticle encapsulated with human serum
albumin, were tested on different cancer cell lines (from ATCC)
including, lung cancer A549, breast cancer MDA-MB-231 and OV 90
ovarian cancer cell lines, as well as on a normal human fibroblast
cell line.
[0182] The drug-induced nanoparticles were at least the same or
more potent at killing the cancer cells, particularly at low drug
concentrations ranging from 0.5 .mu.g/mL to 0.5 ng/mL. No toxicity
to the normal human fibroblast cell line was observed.
[0183] The maximum tolerated dose (MTD) of the drug-induced
nanoparticles was compared to that of Taxol. Over the course of
seven weeks, various doses of Taxol and the paclitaxel-containing
nanoparticles of interest were injected into the tail vein of CD-1
mice. The MTD of the paclitaxel nanoparticles was more than 7-fold
higher than that of Taxol, with no major side effects to the
surviving mice. Also, significant weight loss was observed in mice
receiving Taxol as compared to no weight loss for the cohort that
received the paclitaxel nanoparticles of interest.
[0184] A controlled study using lung cancer A548 cell line and
breast cancer MDA-MB-231 cell line in a xenograft mouse model
revealed that the paclitaxel nanoparticles inhibited tumor growth
in vivo significantly better than did Taxol and Abraxane. A rapid
reduction of tumors in mice receiving the paclitaxel nanoparticles
of interest was observed, as compared to mice receiving Taxol or
Abraxane.
Campothecin Formulation and Nanoparticle Preparation
[0185] Camptothecin (2.1 mg) and polymer (10.5 mg) are dissolved in
a 4:1 v/v chloroform:ethanol mixture. Following thorough mixing,
the solvent is removed to dryness on a rotary evaporator. The
resultant solid mixture is redissolved in 2 mL of saline solution,
mixed, then filtered through a 0.8 um syringe filter. The filtrate
is frozen at -70.degree. C. for at least 2 hours in a
lyophilization vial, then lyophilized overnight (.about.16 hours).
The vial is stoppered and the ready-to-use white powder is stored
at -70.degree. C. The material is reconstituted with 2 mL of water
immediately prior to use.
[0186] Irinotecan, also known as CPT-11, is a synthetic analog of
camptothecin. CPT-11 contains, for example, a bipiperidine
carboxylate group and an ethyl group on the A and B rings, to yield
a compound with greater cytotoxicity than the parent molecule.
[0187] Varying concentrations of CPT-11 and the camptothecin
branched polymer aggregates described above were prepared.
[0188] Two cancer cell lines were maintained in suitable medium
under recognized culture conditions. MCF-7 is a human breast cancer
cell line and H460 is a human epithelial lung cancer cell line.
Those cell lines were exposed to varying concentrations of CPT-11
and concentrations of the camptothecin aggregates based on the
amount of drug. Cell survivability was assessed as described
above.
[0189] FIG. 17 summarizes results obtained with the MCF-7 breast
cancer cell line. It can be seen that cytotoxicity of the cells to
CPT-11 increased with increasing drug concentration. On the other
hand, the camptothecin aggregate was more highly cytotoxic at all
dosages tested. Polymer not associated with drug was not cytotoxic.
Thus, although camptothecin per se does not have the same level of
cytotoxicity as does CPT-11, when aggregated with a branched
polymer, that aggregate was more cytotoxic than CPT-11.
[0190] A similar result was obtained with the H460 lung cancer cell
line. As depicted in FIG. 18, a dose response curve of cytotoxicity
to CPT-11 was observed. However, the camptothecin/branched polymer
aggregates were more cytotoxic than CPT-11 at each concentration
tested.
[0191] All references cited herein are herein incorporated by
reference in entirety.
[0192] It will be appreciated that various changes and
modifications can be made to the teachings herein without departing
from the spirit and scope of the disclosure.
* * * * *