U.S. patent application number 12/041529 was filed with the patent office on 2008-10-09 for particulate drug delivery.
Invention is credited to Jianjun Cheng, Rong Tong.
Application Number | 20080248126 12/041529 |
Document ID | / |
Family ID | 39738725 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080248126 |
Kind Code |
A1 |
Cheng; Jianjun ; et
al. |
October 9, 2008 |
PARTICULATE DRUG DELIVERY
Abstract
Methods for efficient preparation of drug-polymer (or oligomer)
conjugates which are useful in the preparation of particles,
including microparticles and nanoparticles, for delivery of the
drug in vivo for therapeutic applications. The invention
additionally provides certain drug-polymer and drug-oligomer
conjugates which are useful in the preparation of particles for
delivery of the drug in vivo. The invention also provides
nanoparticles of this invention prepared by nanoprecipitation using
drug-polymer/oligomer conjugates of the invention. The drug
conjugates are formed during polymerization of the polymer or
oligomer in which the drug is employed as an initiator of the
polymerization of the monomers which form the polymer and/or
oligomer. More specifically, the drug conjugates are formed by
ring-opening polymerization of cyclic monomers in the presence of
an appropriate ring-opening polymerization catalyst and the
initiator (the drug). The method is particularly useful for
formation of polymer/oligomer conjugates with drugs and other
chemical species containing one or more hydroxyl groups or thiol
groups.
Inventors: |
Cheng; Jianjun; (Champaign,
IL) ; Tong; Rong; (Urbana, IL) |
Correspondence
Address: |
GREENLEE WINNER AND SULLIVAN P C
4875 PEARL EAST CIRCLE, SUITE 200
BOULDER
CO
80301
US
|
Family ID: |
39738725 |
Appl. No.: |
12/041529 |
Filed: |
March 3, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60892834 |
Mar 2, 2007 |
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Current U.S.
Class: |
514/1.1 ;
424/489; 424/490; 514/23; 514/44A; 528/20; 528/332; 528/354;
528/398; 977/773; 977/906 |
Current CPC
Class: |
A61K 31/337 20130101;
A61K 47/605 20170801; A61K 47/6937 20170801; A61K 38/09 20130101;
A61K 38/13 20130101; A61K 47/60 20170801; A61K 47/6803 20170801;
A61K 47/6877 20170801; A61K 47/6811 20170801; C08G 63/823 20130101;
A61K 31/704 20130101; A61K 47/593 20170801; A61K 9/5146 20130101;
A61K 47/59 20170801; A61K 47/6857 20170801; A61K 47/6935 20170801;
A61K 47/6809 20170801; A61K 47/6851 20170801; A61K 47/6927
20170801; C08G 81/00 20130101; A61K 47/6855 20170801; A61K 9/5192
20130101; A61K 31/4355 20130101 |
Class at
Publication: |
424/497 ;
424/489; 424/490; 514/2; 514/23; 514/44; 528/354; 528/398; 528/20;
528/332; 977/906; 977/773 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 38/02 20060101 A61K038/02; A61K 31/70 20060101
A61K031/70; A61K 31/7052 20060101 A61K031/7052; C08G 63/08 20060101
C08G063/08; C08G 79/04 20060101 C08G079/04; C08G 77/04 20060101
C08G077/04; C08G 69/26 20060101 C08G069/26 |
Claims
1. A method for preparing particles for in vivo delivery of a drug
which comprises the steps of: (a) providing a drug the structure of
which comprises one or more hydroxyl or thiol groups; (b)
conducting ring-opening polymerization of one or more cyclic
monomers selected from cyclic esters, cyclic carbonates, cyclic
phosphate, cyclic silicone, cyclic peptides or amino acid
derivative, or cyclic phosphazane, or a combination thereof in
anhydrous, water-miscible solvent in the presence of the drug as a
polymerization initiator and a polymerization catalyst to form a
covalent drug-oligomer or drug-polymer conjugate; and (c) forming
particles comprising the drug-oligomer or drug-polymer conjugate
ranging in size from 2 nanometers to 100 microns.
2. The method of claim 1 wherein the particles are nanoparticles
having particle size ranging from 2 nm to 300 nm.
3. The method of claim 1 wherein the particles are microparticles
ranging in particle size from 0.200 micrometers to 400
micrometers.
4. The method of claim 1 further comprising the step of modifying
the surface of the particle.
5. The method of claim 4 wherein the particle surface is modified
by providing a coating layer of a polymer which may be the same or
different from that of the conjugate.
6. The method of claim 5 wherein the particle surface is modified
with surface pegylation with PEG chain length ranging from PEG 400
to PEG 40,000.
7. The method of claim 6 wherein the surface pegylation is provided
by forming covalent particle-PEG linkages.
8. The method of claim 6 wherein the surface pegylation is provided
through non-covalent interactions using hydrophobic
polymer-b-PEG.
9. The method of claim 4 wherein the surface of the particle formed
is modified with a hydrophilic or hydrophobic surface modifier.
10. The method of claim 4 wherein the surface of the particle
formed is modified with an amphiphilic polymer.
11. The method of claim 10 wherein the amphiphilic polymer
comprises PEG.
12. The method of claim 10 wherein the amphiphilic polymer is a
copolymer comprising PEG.
13. The method of claim 9 wherein the surface of the particle
formed is modified by covalent or non-covalent attachment to one or
more of a peptide, protein, saccharide, carbohydrate, nucleic acid
or a combination thereof.
14. The method of claim 1 wherein the terminal groups of the
polymer or oligomer are selected from the group consisting of
hydroxyl, thiol, amine, azide, alkyne, alkene, ketone, phenol,
halides, imidazole, guanidinium, carboxylate, or phosphate
groups.
15. The method of claim 14 wherein the surface of the particle
formed is modified by coating or conjugation of one or more
targeting ligands.
16. The method of claim 1 wherein the particles have a drug
concentration gradient.
17. The method of claim 1 wherein the particles have a multilayer
structure with different drug concentrations or different types of
drugs in different layers.
18. The method of claim 17 wherein at least one layer of the
particles is formed from the drug-oligomer or drug-polymer
conjugate.
19. The method of claim 1 wherein the drug is a drug selected from
the drugs in FIG. 13.
20. The method of claim 1 wherein the drug is a drug selected from
Darunavir (TMC-114), Tipranavir (TPV), Saquinavir (SQV), Ritonavir
(RTV), Indinavir, Nelfinavir (NFV), Amprenavir (APV), Lopinavir
(ABT-378), Atazanavir (ATV), Vinorelbine bitartrate, fulvestrant,
Sarcodictyins, camptothecins, Vinblastine, bryostatin 1,
(+)-Cylindricine, (+)-Lactacystin, Aeruginosin 298-A,
(+)-Fostriecin, Garsubellin A/Hyperforin, (S)-Oxybutynin,
Epothilone A, Zidovudine (AZT), Lamivudine (3TC), Didanosine (ddI),
Abacavir (ABC), Emtricitabine (FTC), bamethane, ethamivan,
hexachlorophene, salicylanilide, pyrocatechin, thymol, pentazocine,
phloroglucinol, eugenol, niclosamide, terbutaline, dopamine,
methyldopa, norepinephrine, eugenol, .alpha.-naphthol, polybasic
phenols, adrenaline, dopamine, phenylephrine, metaraminol,
fenoterol, bithionol, alpha-tocopherol, isoprenaline, adrenaline,
norepiniphrine, salbutamol, fenoterol, bithionol, chlorogenic
acid/esters, captopril, amoxicillin, betaxolol, masoprocol,
genistein, daidzein, daidzin, acetylglycitin, equol, glycitein,
iodoresiniferatoxin, SB202190, or tyrphostin SU1498.
21. The method of claim 1 wherein the nanoparticles formed range in
size on average from 2 nm to 400 .mu.m.
22. The method of claim 1 wherein the catalyst is an organometallic
catalyst or an organo catalyst of a ring-opening polymerization
reaction.
23. The method of claim 1 wherein the polymerization reaction is
performed in a solvent selected from THF, acetone, methylene
chloride, chloroform, dimethylformamide, DMSO, acetonitrile or
mixtures thereof.
24. The method of claim 1 wherein nanoparticles are formed by
combining a solution of the covalent drug-polymer conjugate or the
drug-oligomer conjugate in a water-miscible solvent with an excess
of water.
25. The method of claim 1 wherein the molar ratio of cyclic monomer
to drug initiator ranges from 5000/1 to 2/1.
26. The method of claim 1 wherein the drug is a hydroxyl-containing
small organic molecule.
27. The method of claim 1 wherein the drug is a macromolecule.
28. The method of claim 1 wherein the drug is a peptide, saccharide
or nucleic acid.
29. The method of claim 1 wherein the cyclic monomer is a cyclic
ester, cyclic carbonate or a combination thereof.
30. The method of claim 1 wherein the catalyst is an orgaometallic
catalyst.
31. A covalent drug-oligomer or drug-polymer conjugate prepared by
polymerizing one or more cyclic monomers selected from the group
consisting of cyclic esters, cyclic carbonates, cyclic phosphates,
cyclic siloxanes, cyclic peptides or amino acid derivative, or
cyclic phosphazanes, in the presence of a drug the structure of
which comprises one or more hydroxyl groups and a ring-opening
polymerization catalyst wherein the drug comprising the one or more
hydroxyl groups is the initiator of the polymerization
reaction.
32. The covalent drug-polymer conjugate of claim 31 wherein the
oligomer comprises 5000 or fewer repeating units of the ring-opened
monomer.
33. The covalent drug-polymer conjugate of claim 31 wherein the
oligomer comprises 500 or fewer repeating units of the ring-opened
monomer.
34. The covalent drug-oligomer conjugate of claim 31 wherein the
oligomer comprises 50 or fewer repeating units of the ring-opened
monomer.
35. The covalent drug-oligomer conjugate of claim 31 wherein the
oligomer comprises 20 or fewer repeating units of the ring-opened
monomer.
36. The covalent drug-oligomer conjugate of claim 31 wherein the
oligomer comprises 10 or fewer repeating units of the ring-opened
monomer.
37. A pharmaceutical composition comprising particles made by the
method of claim 1.
38. A pharmaceutical composition comprising particles made from a
covalent drug-oligomer or drug-polymer conjugate of claim 31.
39. A nanoparticle comprising a core/shell structure or a multiple
layer structure wherein at least one of the core or shell or one of
the layers is formed from a drug-polymer or a drug-oligomer
conjugate which is prepared by conducting ring-opening
polymerization of one or more cyclic monomers selected from cyclic
esters, cyclic carbonates, cyclic phosphate, cyclic silicone,
cyclic peptides or amino acid derivative, or cyclic phosphazane, or
a combination thereof in an anhydrous, water-miscible solvent in
the presence of the drug as a polymerization initiator and a
polymerization catalyst to form the drug-oligomer or drug-polymer
conjugate.
40. A method for delivery of a drug to an individual in need
thereof which comprises administering to the individual
nanoparticles of claim 39 and which comprise the drug covalently
conjugated to an oligomer or polymer comprising ring-opened monomer
repeating units.
41. The method of claim 40 wherein the monomers are selected from
lactides, glycolides or a combination thereof.
42. A method for delivery of a drug to an individual in need
thereof which comprises administering to the individual
nanoparticles prepared by the method of claim 1 and which comprise
the drug covalently conjugated to an oligomer or polymer comprising
ring-opened monomer repeating units.
43. A method for making a medicament comprising a drug whose
structure comprises one or more hydroxyl groups or thiol groups
which comprises covalently attaching the drug to an oligomer or
polymer formed by rein-opening polymerization by introducing the
drug into the ring-opening polymerization reaction to function
therein as the initiator of polymerization.
44. A method for preparing particles for in vivo delivery of a
chemical species which has at least one hydroxyl group or thiol
group which comprises the steps of: (a) conducting ring-opening
polymerization of one or more cyclic monomers selected from cyclic
esters, cyclic carbonates, cyclic phosphate, cyclic silicone,
cyclic peptides or amino acid derivative, or cyclic phosphazane, or
a combination thereof in anhydrous, water-miscible solvent in the
presence of the chemical species having at least one hydroxyl group
as a polymerization initiator and a polymerization catalyst to form
a covalent drug-oligomer or drug-polymer conjugate; and (c) forming
particles comprising the drug-oligomer or drug-polymer conjugate
ranging in size from 2 nanometers to 100 microns.
45. A covalent oligomer or polymer conjugate prepared by
polymerizing one or more cyclic monomers selected from the group
consisting of cyclic esters, cyclic carbonates, cyclic phosphates,
cyclic siloxanes, cyclic peptides or amino acid derivative, or
cyclic phosphazanes, in the presence of a chemical species the
structure of which comprises one or more hydroxyl groups and a
ring-opening polymerization catalyst wherein the drug comprising
the one or more hydroxyl groups is the initiator of the
polymerization reaction.
46. The conjugate of claim 45 wherein the chemical species is
selected from the groups consisting of a diagnostic reagent, a
peptide, a saccharide, a carbonhydrate, an inorganic species, a
contrast agent, a vitamin, a nutrient, a nucleic acid, an RNA
molecule, an siRNA, or a DNA molecule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application 60/892,834, filed Mar. 2, 2007, which is incorporated
by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] Polymer nanoparticles (NPs) play an important role in drug
delivery and are particularly useful for delivery of chemotherapy
drugs. For clinical applications, the control of nanoparticle size
and surface morphology are important. Other aspects of the design
of particulate systems can also be important for the use of
nanoparticles as delivery systems in vivo. It is preferred that
drug loading in the polymeric nanoparticle is reasonable high for
improved efficacy. This is particularly important for enhanced
effectiveness of nanoparticles in cancer therapy.sup.1-3
[0003] High drug loading.sup.4,5 decreases manufacturing cost and
increases patient compliance by reducing the dose needed for each
administration. In addition, drug molecules in nanoparticle
delivery vehicles preferably remain substantially encapsulated in
the polymeric nanoparticles on administration to a patient to be
released in a sustained manner over time or after they accumulate
at a desired location. More specifically for nanoparticle
applications to cancer therapy, it is important that anticancer
agents remain encapsulated with little or no drug release while in
the vasculature and release the anticancer agent only after the
nanoparticles extravasate to tumor tissues.
[0004] Well-controlled drug release has been realized only in a
limited number of drug delivery systems, most of which are
liposomes..sup.6 Currently, there are about 10 liposomal delivery
vehicles approved for clinical applications of which only one,
Abraxane, an albumin bound paclitaxel nanoparticle with size
.about.130 nm, is a polymeric nanoparticulate delivery
vehicle..sup.6,7 Abraxane,.sup.8, 9 appears to contain a large
quantity of lipids on the surface of albumin nanoparticles which
gives them liposome-like properties in regulating drug release. To
date no polyester based nanoencapsulates are approved for clinical
cancer treatment.
[0005] In a recent study using poly(ethylene
glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) to nanoencapsulate
docetaxel for in vivo prostate cancer treatment (Farokhzad, O. C.;
Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P. W.;
Richie, J. P.; Langer, R. Proc. Nat'l Acad. Sci. (USA) 2006, 103,
6315-6320), difficulties were experienced in controlling
formulation parameters such as drug loading and encapsulation
efficiency. The encapsulation efficiency, which depends on various
parameters, including solvents, type of polymers, polymer molecular
weights, and the drugs to be encapsulated, varied from batch to
batch and was usually less than 80%. Drug loading was also
typically lower than 10%. In many cases, only 1% of drug loading
could be achieved. NPs with more than 5% of drug loading sometimes
contained undesired large aggregates (>1 micron), which was
presumably due to the aggregation of the non-encapsulated drug
molecules. Particles with mixed sizes and wide distributions can
lead to complex biodistribution and pharmacokinetic responses in
vivo.
[0006] In liposome delivery systems, drug molecules are
encapsulated in the core of the liposome and are, thus, separated
from the external environment by lipid bi-layers which prevent
leakage of encapsulated drug molecules. In contrast, polymer
nanoencapsulates (polymeric nanoparticles) have no such regulating
mechanism to prevent the unwanted leaking of therapeutic molecules
during circulation. Significant burst release effects are one of
the greatest challenges to overcome in the application of polymeric
nanoparticles in vivo for drug delivery. In a vehicle with
significant burst release effect, poorly encapsulated drug
molecules on or near the surface of nanoparticles can quickly
diffuse into solution and may lead to significant toxicity in
vivo..sup.10 Burst release is especially severe when drug loading
exceeds the encapsulation threshold of the polymer where there can
be a significant amount of drug molecules precipitated on the
surface of the nanoparticle.
[0007] Nanoencapsulates (NE) usually display a biphasic drug
release pattern.sup.10-12 with as high as 40-80% of the
encapsulated drug molecules burst released during the first several
or tens of hours..sup.10 After the first 24 to 48 hours, drug
release becomes significantly slower due to the increased diffusion
barrier for drug molecules buried more deeply in polymer
nanoparticles. When these semi- or even completely empty
nanoparticles eventually arrive and accumulate at the site where
they are needed (e.g., tumor tissue), they usually have little or
no remaining therapeutic efficacy..sup.6, 13
[0008] It is extremely difficult to achieve high drug loading with
high encapsulation efficiency in polymeric nanoencapsulates. The
encapsulation efficiency not only depends on the type, molecular
weight and properties of the polymers used, but is also
significantly affected by the chemical and physical properties of
therapeutic molecules. For example, lower molecular weight polymers
tend to exhibit lower encapsulation efficiency than higher
molecular weight polymers. Hydrophilic molecules (e.g.,
doxorubicin) cannot be readily encapsulated into polymeric
nanoparticles (Grovender T. et al. (1999) J. Controlled Release
57(2) 171-185). In all nanoencapsulates so far developed, drug
loading (the weight percentage of drug in polymer nanoparticles)
and encapsulation efficiency (percentage of drug encapsulated
relative to total amount of drugs applied) vary dramatically from
system to system and from batch to batch. For hydrophobic small
molecules such as paclitaxel (Ptxl) or docetaxel (Dtxl), it is
common that nanoparticle loading is in a range of 1 to 5 wt % and
encapsulation efficiency varies from .about.20- to 80%..sup.10,
14
[0009] Another problem encountered in nanoparticle drug
encapsulation is undesirable particle heterogeneity.
Nanoprecipitation of polymer and drugs, such as chemotherapeutics,
frequently gives multimodal distributions as measured by dynamic
light scattering, ranging from .about.100 nm to 1 .mu.m or higher.
Particle heterogeneity may result because encapsulation involves
distinct chemical species, a polymer and a drug molecule, with
distinct molecular weights, flexibility and rigidity,
hydrophobicity and tendencies toward forming crystals. Therefore it
is likely the polymer and the drug molecule would tend to
self-aggregate during nanoprecipitation leading to particle
heterogeneity.
[0010] Nanoparticle materials exhibiting multimodal distributions
are usually treated as having a different degree of aggregation of
small nanoparticles with identical composition. However, this
assumption may not always be correct. In a recent investigation on
the effect of docetaxel loading at 1%, 5% and 10% on resulting
PEG-b-PLGA nanoparticle size distributions (Cheng, J. et al.
Formulation of functionalized PLGA-PEG nanoparticles for in vivo
targeted drug delivery. Biomaterials 28, 869-76 (2007)),
polydispersity of the particle preparations increased with
docetaxel concentration from 0.154 for 1% loading to 0.203 for 5%
loading and 0.212 for 10% loading. The size distribution of the
nanoparticles exhibited a biphasic trend with a smaller diameter
particle distribution accompanied by a distribution of larger
diameter particles. The distribution corresponding to the smaller
particles did not shift with the increase of drug concentration.
The larger diameter locus of the two size distributions shifted
higher as the drug loading increased (the size increasing from
.about.300 nm to .about.1200 nm). Since the only difference between
these formulations is the amount of drug loading, a significant
amount of the nanoparticles formed may be due to aggregation of
unencapsulated docetaxel due to its poor water solubility. In this
work and that of others (Avgoustakis, K. et al. PLGA-mPEG
nanoparticles of cisplatin: in vitro nanoparticle degradation, in
vitro drug release and in vivo drug residence in blood properties.
J. Controlled Release 79, 123-135 (2002)) on nanoprecipitation
using polylactide and docetaxel, biphasic particle distributions
were almost always observed.
[0011] It is desirable to develop a methodology to circumvent these
difficulties, which will provide NPs with batch-to-batch
consistency in encapsulation efficiency and drug loading. This
invention provides a simple, one-step strategy for the preparation
of drug-polymer (and drug-oligomer) conjugates which can be formed
into nanoparticles with 100% encapsulation efficiency and
predetermined drug loading. The nanoparticles formed by the methods
herein employing drug conjugates are call nanoconjugates herein to
distinguish over nanoencapsulates (NE). Further, the method of this
invention can be broadly applied to provide polymer and oligomer
conjugates of a variety of useful chemical species (bioactive
species, drugs, reagents, diagnostics, contrast agents, reporter
molecules, dyes, etc.) for the preparation of particulate delivery
systems.
SUMMARY OF THE INVENTION
[0012] In one embodiment, this invention provides a one-step method
for efficient preparation of certain drug-polymer (or oligomer)
conjugates which are useful in the preparation of particles,
including microparticles and nanoparticles, for delivery of the
drug in vivo for therapeutic applications. The invention
additionally provides certain drug-polymer and drug-oligomer
conjugates which are useful in the preparation of particles for
delivery of the drug in vivo. The invention also provides a method
of making particulate drug delivery systems or vehicles employing
the drug-polymer or drug-oligomer conjugates of this invention. The
polymer and oligomer conjugates of this invention can be employed
in any art-known method for the preparation of particles from
polymers or oligomers. The methods herein are particularly useful
for the preparation of nanoparticles for drug delivery and more
particularly are useful for the preparation of nanoparticles for
chemotherapy applications. Nanoparticles of this invention are, for
example, prepared by nanoprecipitation methods from the polymer
and/or oligomer conjugates described in this invention.
[0013] In specific embodiments, the invention provides particles
containing a selected drug optionally for sustained or targeted
drug delivery. In specific embodiments, a selected drug is
substantially (90% or more by weight of the drug) covalently bound
to polymer or oligomer in the particle. The particles are prepared,
for example, by known methods from solutions containing
drug-polymer and/or drug-oligomer conjugates. The drug conjugates
of this invention are formed during polymerization of the polymer
or oligomer in which the drug is employed as an initiator of the
polymerization of the monomers which form the polymer and/or
oligomer. More specifically, the drug conjugates are formed by
ring-opening polymerization of cyclic monomers in the presence of
an appropriate ring-opening polymerization catalyst and the
initiator (the drug).
[0014] In specific embodiments, the drug-polymer or drug-oligomer
conjugates of this invention are employed to make various types of
particles useful for drug delivery containing the drug and ranging
generally in size from about 2 nm to about 500 microns. In other
more specific embodiments, the drug-polymer or drug-oligomer
conjugates of this invention are employed to make microparticles
containing the drug and ranging generally in size from about 500 nm
to about 100 microns. In other embodiments, the drug-polymer or
drug-oligomer conjugates of this invention are employed to make
nanoparticles containing the drug and ranging generally in size
from about 55 nm to about 600 nm. In other embodiments, the
drug-polymer or drug-oligomer conjugates of this invention are
employed to make particles containing the drug and ranging
generally in size from about 2 nm to about 100 nm. In other
embodiments, the drug-polymer or drug-oligomer conjugates of this
invention are employed to make particles containing the drug and
ranging generally in size from about 200 nm to about 800 nm. In
other embodiments, the drug-polymer or drug-oligomer conjugates of
this invention are employed to make particles containing the drug
and ranging generally in size from about 1 micron to about 500
micron.
[0015] In a specific embodiment, the methods of this invention can
be employed to make nanoparticles in the 20-60 nm size range. Such
nanoparticles can be made, for example by known micellation methods
from polymer or oligomer conjugates as described herein followed by
further reaction with a PEG-capping agent, for example
PEG-isocyanate.
[0016] In another specific embodiment, the methods of this
invention can be employed to make nanoparticles in the 1-20 nm
range which are particularly useful for delivery to cells. Such
nanoparticles are formed employing cyclic AB2 type monomers or
mixtures of such monomers with other cyclic esters and carbonate
monomers described herein above. AB2 type monomers polymerize in
the methods herein to form hyperbranched or dendritic structures
conjugated to a selected drug molecule. Particles formed directly
by polymerization of the AB2 type monomers can be used for drug
delivery. Alternatively, these particles can be subjected to
surface treatments as discussed herein below.
[0017] The method of this invention is useful for forming polymer
or oligomer conjugates with any small molecule drug (i.e., a small
molecule drug is a non-peptide, non-sugar and non-nucleic
acid-based drug) which contains at least one functional group which
can function for initiation of the ring-opening polymerization
reaction, e.g. a hydroxyl group or a thiol group. The drug may
contain, but need not contain, a plurality of such polymerization
initiation groups, e.g., a plurality of hydroxyl groups or thiol
groups. In preferred embodiments, the drug contains only one of
such polymerization groups. The hydroxyl groups may be primary,
secondary or tertiary hydroxyl groups. Similarly, the thiol groups
may be primary, secondary or tertiary thiol groups. The hydroxyl
group may also be a phenolic hydroxyl group. In specific
embodiments, the drug contains one or more non-phenolic hydroxyl
groups. In specific embodiments, the drug contains one or more
non-phenolic hydroxyl groups which are primary or secondary
hydroxyl groups. In specific embodiments, the drug contains a
single non-phenolic hydroxyl group. In specific embodiments, the
drug contains a single primary or secondary hydroxyl group.
Exemplary drugs which can be employed in the methods herein are
listed and illustrated in FIGS. 8 and 13 and additional drugs are
listed below.
[0018] In specific embodiments, the drug is hydrophilic and in
related embodiments, the drug is water-soluble (e.g., exhibiting
solubility in water in the range of mg/mL). In other specific
embodiments, the drug is hydrophobic and in related embodiments,
the drug is not water-soluble or exhibits low water solubility
(e.g., exhibiting solubility in water in the range of micrograms
per mL or less).
[0019] In specific embodiments, the drug which is conjugated to the
polymer or oligomer in the methods herein is a drug that is an
anticancer agent or that is useful in chemotherapy. In specific
embodiments, the drug is a taxane. In other specific embodiments,
the drug is an anticancer agent of the anthracyclin family. In
other embodiments, the drug is a protease inhibitor. In other
specific embodiments, the drug is an inhibitor of reverse
transcriptase. In other specific embodiments, the drug is an
antiviral agent. In other specific embodiments, the drug is an
antifungal agent. In other specific embodiments, the drug is a
phenolic drug, i.e., having one or more phenolic hydroxyl groups.
In other specific embodiments, the drug is a thiol drug, i.e.,
having one or more thiol groups.
[0020] The method of this invention is also useful for delivery of
drugs which are peptides, proteins, sugars and/or nucleic acid (DNA
or RNA). In each case, the drug must contain at least one
functional group that can function as an initiator in the
ring-opening polymerization reaction, e.g., at least one hydroxyl
or one thiol group.
[0021] The method of this invention can be more broadly applied to
any molecule or other chemical species (including synthetic, or
naturally-occurring molecules and organic or inorganic species)
which contains at least one functional group which can function as
an initiator in a ring-opening polymerization reaction (e.g., a
hydroxyl group or a thiol group) and which one wishes to administer
or deliver in vivo using a particulate delivery system such as a
microparticle or a nanoparticle. The method may be applied to form
polymer or oligomer conjugates with any such useful chemical
species including without limitation, reagents for diagnostic
methods, nutrients or vitamins (which may also be considered
drugs), or reporter molecules (e.g. radiolabeled or fluorescently
labeled molecules). The chemical species to be conjugated to the
polymer or oligomer may be hydrophilic, hydrophobic, water-soluble
or water-insoluble. The chemical species may contain a plurality of
hydroxyl groups or thiol groups which may be primary, secondary or
tertiary hydroxyl groups and which may be phenolic hydroxyl groups.
In specific embodiments, the hydroxyl groups are primary or
secondary hydroxyl groups. In specific embodiments, the hydroxyl
groups are phenolic hydroxyl groups. In specific embodiments, the
thiol groups are primary or secondary hydroxyl groups. In specific
embodiments, the chemical species is a chemical species other than
a saccharide. In specific embodiments, the chemical species is a
chemical species other than a carbohydrate.
[0022] In specific embodiments of all of the above-listed cyclic
monomer, one or both of Y.sub.1 and Y.sub.2 are haloalkyl groups,
particularly fluoroalkyl groups.
[0023] The scope of the invention as described and claimed
encompasses the use of racemic forms of the cyclic monomers as well
as the individual enantiomers and non-racemic mixtures thereof.
[0024] The methods herein employ any appropriate ring-opening
polymerization catalyst which may be a metal-containing catalyst or
an organocatalyst. In specific embodiments, the catalysts are
selected from Mg(II) or Zn(II) catalysts. In other specific
embodiments, 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD),
N-methyl-TBD (MTBD), or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
are employed as organocatalysts.
[0025] The molar ratio of combined monomer(s) to initiator(s)
ranges most generally from 2:1 to 5000:1, and more specifically
from 5:1 to 200:1 and yet more specifically from 10:1 to 100:1. In
the methods herein two or more different cyclic monomers may be
combined in the polymerization reaction to form copolymer
conjugates. In the methods herein two or more different cyclic
monomers may be sequentially added to the polymerization reaction
to form block co-polymer conjugates. Two or more chemical species
each having at least one hydroxyl or thiol group can be combined in
the methods herein to provide a mixture of polymer or oligomer
conjugates with the different chemical species. For example, two
different drugs each having at least one hydroxyl group or thiol
group may be combined in the polymerization method herein to form a
mixture of polymer or oligomer drug conjugates. It can be
beneficial, for example, to combine two or more drugs exhibiting
different mechanisms of action for treatment of the same or related
disorders, diseases or conditions.
[0026] In specific embodiments, particles, particularly
nanoparticles, formed by the methods herein can exhibit drug
loading (or more generally loading of the selected chemical
species) that is 20% or more, 30% or more, 40% or more, or 50% or
more.
[0027] In specific embodiments, particles, particularly
nanoparticles, formed by the methods herein can exhibit long
circulation lifetimes useful for effective in vivo delivery. This
is particularly the case when the particles are surface modified
employing methods described herein or employing methods that are
known in the art. In specific embodiments, particles, particularly
nanoparticles, formed by the methods herein can exhibit stability
in salt solutions.
[0028] In the methods herein, formation of polymer or oligomer
conjugates can be combined with any known method for the formation
of particles, including nanoprecipitation, micellation, emulsion
and double emulsion methods.
[0029] The invention also provides certain polymer or oligomer
conjugates which are prepared by the polymerization methods herein.
These conjugates can in general be those with any chemical species
that it is desired to deliver in a particulate delivery system and
particularly are drugs and most particularly are anticancer or
chemotherapeutic drugs. In specific embodiments, the conjugates are
those in which the polymer of the conjugate on average has 100 or
fewer monomer units. In other embodiments, the conjugates are those
in which the polymer of the conjugate has on average 75, 50, or 25
monomer units. In specific embodiments, the conjugates are those in
which the polymer has weight average molecular weight of 5000 or
less, 2500 or less, 1500 or less, or 1000 or less. In specific
embodiments, the invention provides certain polymer or oligomer
conjugates prepared by the polymerization methods herein and in
which the chemical species of the conjugate is conjugated or bonded
to only one polymer or oligomer. In specific embodiments, the
invention provides certain polymer or oligomer conjugates prepared
by the polymerization methods herein and in which the chemical
species of the conjugate is conjugated or bonded to only one
polymer or oligomer and at only one site in the chemical
species.
[0030] In specific embodiments, the invention provides polymer or
oligomer conjugates to hydrophilic chemical species, and in
particular to hydrophilic drugs. In other specific embodiments, a
hydrophobic chemical species, particularly a hydrophobic drug is
conjugated to the polymer or oligomer.
[0031] The invention further provides particles, including
microparticles and nanoparticles, comprising the polymer conjugates
or oligomer conjugates of this invention which are useful for in
vivo delivery of selected chemical species, more particularly one
or more drugs and most particularly one or more anticancer or
chemotherapeutic agents. In specific embodiments, the particles,
including microparticles or nanoparticles are surface-modified by
any means known in the art, for example, with one or more
antibodies, with one or more nucleic acid molecules, e.g.,
aptamers, with one or more peptides or proteins, e.g., enzymes,
with one or more polymers or oligomers, e.g., amphiphilic polymers,
particularly amphiphilic polymers containing PEG.
Surface-modification of particles as is known in the art can
facilitate targeting of particles to certain tissue, can facilitate
entry of particles into cells or can enhance stability of the
particle. For example, nanoparticles formed from polyesters,
polycarbonates or mixtures thereof can be coated with hydrophilic
polymers such as PEG or amphiphilic polymers containing PEG to
enhance circulation lifetime of the nanoparticle.
[0032] In additional embodiments, the invention provides particles
having a core/shell structure or having a multiple layer structure
in which at least one of the core or shell or one of the multiple
layers is a layer which is formed from the drug (or other chemical
species)-polymer/oligomer conjugates of this invention. In
particular, the invention relates to nanoparticles having a
core/shell structures in which the core or shell is formed from a
polymer/oligomer conjugate of this invention. More specifically,
nanoparticles can be formed with a core that is formed from a first
polymer/oligomer conjugate and a shell that is formed from (1) a
polymer, e.g., a hydrophilic polymer or an amphiphilic polymer or
(2) a second polymer/oligomer conjugate of this invention. In
specific embodiments, the first and second polymer/oligomer
conjugates can be selected from those of a taxane, an anthracycline
antibiotic, or a Shh antagonist which has a functional group, such
as a hydroxyl or thiol group that can function for polymerization
initiation as described herein. In more specific embodiments, the
first and second polymer/oligomer conjugates can be selected from
those of Ptxl, Dtxl, Doxo, cyclopamine, or camptothecin. The
invention specifically provides multiple layer nanoparticles
containing three or more different layers wherein at least one
layer is formed from a polymer/oligomer conjugate of this
invention. Nanoparticles include those having three, four or five
layers. Nanoparticles include those in which all layers are formed
from polymer/oligomer conjugates of this invention. Nanoparticles
include those in which at least one layer is formed from a
polymer/oligomer conjugate of this invention and at least one other
layer is formed from a polymer (non-conjugated polymer) such as a
hydrophilic, hydrophobic or amphiphilic polymer. In specific
embodiments, nanoparticles include those in which at least one
layer is formed from a polymer/oligomer conjugate of this invention
and at least one other layer is formed from an amphiphilic polymer
comprising PEG.
[0033] The invention additionally provides methods for making a
medicament employing the polymer or oligomer conjugates of this
invention as well as the medicaments made thereby. Medicaments are
particles, particularly nanoparticles, formed from the conjugates
of this invention.
[0034] The invention further provides kits for carrying out the
polymerization reactions herein to form polymer or oligomer
conjugates with a selected chemical species having at least one
hydroxyl group. The kits comprise one or more containers which in
turn comprise one or more cyclic monomers and one or more
ring-opening polymerization catalysts and optionally include
instructions for carrying out the polymerization reaction,
instructions for making particles, one or more reagents or
instructions for surface modification of particles, one or more
solvents for carrying out the polymerization or for making
particles, one or more control initiators, additional receptacles
for carrying out the reaction, for forming particles or for
carrying out surface modification. In specific embodiments, kits
herein comprise a plurality of different cyclic monomers useful for
making conjugates with different oligomers or polymers. In other
embodiments, kits herein can further contain one or more different
chemical species having at least one hydroxyl group for forming
conjugates.
[0035] Additional embodiments of the invention will be apparent on
review of the following detailed description, examples and
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIGS. 1A and 1B are schematic illustrations of the formation
of nanoencapsulates (prior art) and nanoconjugates of this
invention, respectively. The figures illustrate the structural
differences between the nanoencapsulates and the
nanoconjugates.
[0037] FIG. 2A is a schematic illustration of lactide
polymerization of the invention exemplified for drug initiation of
polymerization in the presence of catalyst for the preparation of
high loading, 100% incorporation efficiency, controlled-releasing
nanoconjugates for in vivo application.
[0038] FIG. 2B provides a specific example of synthesis of
exemplified pegylated pacitaxel-containing nanoconjugates by Ptxl
initiated polymerization of lactide in the presence of
(BDI)MN(TMS).sub.2 (where M=Mg or Zn), followed by
nanoprecipitation and non-covalent surface pegylation with
PLGA-mPEG. The polymerization is believed to be initiated through
formation of a (BDI)M-oxide with Ptxl.
[0039] FIG. 3 is a graph of the release kinetics at 37 C in
1.times.PBS of Pxtl from Pxtl-LA nanoconjugates: Pxtl-LA.sub.25 NC
and Pxtl-LA.sub.50 NC (drug loading indicated in figure). Also
included in the graph for comparison is the release kinetics of a
Pxtl/PLA nanoencapsulate (NE) prepared by nanoprecipitating a
mixture of Ptxl and PLA (Pxtl/PLA (wt/wt)=1/12).
[0040] FIG. 4 is a graph showing toxicity evaluation of
Ptxl-LA.sub.50 NC, Ptxl-LA.sub.25 NC, Ptxl-LA.sub.10 NC and Pxtl
using the MTT assay in PC-3 cells after 24 h incubation. The "*"
indicates significance at 95% confidence interval.
[0041] FIG. 5 is a graph showing changes in particle size when
PLGA-mPEG.sub.5k is added to Pxt-LA.sub.200 NC. Particle size
increases linearly as a function of the weight ratio of amphiphilic
copolymer to drug-polymer conjugate.
[0042] FIG. 6 is a graph illustrating the stability of
Ptxl-LA.sub.200 NC in PBS at 37 C before (.box-solid.) and after
treatment with PLGA-mPEG.sub.5k (.diamond-solid.) or mPEG.sub.5k (
).
[0043] FIGS. 7A-D are graphs showing the results of MTT studies of
Ptxl-LA, Dtxl-LA, CPT-LA and Doxo-LA NCs cytotoxicity on PC-3
prostate cancer cells.
[0044] FIG. 8 provides the chemical formulas for several drugs or
other chemical species (e.g., Cy5 reported dye) which have been
incorporated into NCs employing the methods herein.
[0045] FIG. 9 is a schematic illustration of a method for
preparation of dendritic nanoparticles, which are believed to be
unimolecular dendritic particles containing one therapeutic in each
nanoparticle. The polymerization method is similar to that
described. These dendritic particles give <20 nm particle size,
a range unachievable using other strategy.
[0046] FIG. 10A is a graph illustrating the change in size of
nanoparticles (nm) during a multidrug layer-by-layer precipitation
process. In this process, a nanoconjugate formed by
nanoprecipitation of a drug-polymer conjugate of this invention is
treated with a second drug-polymer conjugate, under
nanoprecipitation conditions, to form a shell or second layer of
the second drug polymer in the nanoconjugates. The figure provides
a size distribution plot of nanoparticle size change on the
formation of a nanoparticle with a Dtxl-LA.sub.100 core and a
Doxo-LA.sub.100 shell or second layer.
[0047] FIG. 10 B is a plot of nanoparticle size (nm) as a function
of the amount of shell-forming (second drug-polymer) conjugate on
nanoprecipitation of a second drug polymer conjugate onto
nanoparticles (NCs) formed from a first drug polymer conjugate. The
plot compares size change as a function of the amount of the second
drug-polymer conjugate added. In one case, the second drug
conjugate is the same as the first drug conjugate and the plot
illustrates the effect of adding increasing amounts of the same
drug-polymer conjugate.
[0048] FIGS. 11A and 11B provide the results of luciferase assays
of NC containing cylcopamine added to Shh-Light 2 cells bearing
luciferase-encoded Gli-1gene. FIG. 11A shows results with addition
of CA-LA.sub.10 and CA-LA.sub.25 where loading is indicated in
parentheses. The figure shows that the EC.sub.50 of the cyclopamine
NCs are significantly lower than that of free cyclopamine,
demonstrating that the nanoconjugate allowed delivery, concentrated
accumulation and release of cyclopamine in the targeted cells. FIG.
11B shows the results of culturing the Shh-Light 2 cells with CA
NCs along with NEs containing purmorphamine which is a
Shh-agonist.
[0049] FIG. 12 provides the actual MW and PDI of Ptxl-LA
conjugates, measured by GPC, formed using different catalysts
(shown in the figure) in the LA polymerization synthesis of the
conjugates.
[0050] FIG. 13 is a partial list of drugs that are useful in the
methods of the invention. The list includes structures.
DETAILED DESCRIPTION OF INVENTION
[0051] The present invention is based at least in part on the
discovery that polymer and oligomer conjugates with drugs and other
chemical species that can function as an initiator of ring-opening
polymerization can be readily prepared in a single step
polymerization synthesis in which the chemical species initiator is
combined with one or more cyclic monomers and a ring-opening
polymerization catalyst. FIG. 2 schematically illustrates the
methods of this invention (A) and illustrates a specific example of
nanoconjugate formation using paclitaxel conjugated to PLA.
Further, it has been discovered that the polymer and oligomer
conjugates thus formed are useful in the preparation of particles,
including microparticles and nanoparticles, having particle sizes
that are useful for the delivery of the chemical species in vivo.
As specifically illustrated in FIG. 2, nanoprecipitation methods
can be used to form nanoparticles (nanoconjugates) containing the
conjugates of this invention. As further illustrated in FIG. 2
nanoparticle nanoconjugates can be treated with PEG to peglyate the
surface of the nanoparticle.
[0052] Polymer and oligomer conjugates formed by the methods herein
are distinguishable from conjugates formed by conjugation of a
chemical species, particularly a drug, with a pre-formed polymer.
The conjugates formed can exhibit polymer average molecular weight
much lower than pre-formed polymers. The conjugates formed can
exhibit polydispersity must lower than pre-formed polymers. For
example, polymer conjugates of this invention can exhibit
polydispersities of 1.5 or less, 1.3 or less and 1.2 or less. In
general the conjugates formed by the methods herein will be more
uniform in polymer length than those formed by conjugation of a
chemical species with a pre-formed polymer.
[0053] Nanoconjugates (NCs) formed from nanoprecipitation of the
polymer or oligomer conjugates of this invention are
distinguishable from nanoencapsulates (NEs) with respect to drug
loading, drug encapsulation, drug release, particle distribution as
well as ease of manufacture, as illustrated in FIG. 1. NEs exhibit
low to medium drug loading (1-5 wt %), which it is not possible to
predetermine and which can vary from batch to batch. NCs exhibit
predefined drug loading levels with much higher batch to batch
consistency. NEs exhibit uncontrollable encapsulation efficiency
(ranging 10-80%), which vary from batch-to-batch, and
system-to-system and which are unable to encapsulate hydrophilic
drugs. NC's exhibit circa 100% encapsulation efficiency, with
little or no batch-to-batch and system-to-system variation and can
be formed with both hydrophilic and hydrophobic drugs. NEs exhibit
significant burst release with 40-80% release in the first 24
hours. NCs exhibit little or no burst release of drug and provide
for adjustable and controllable release of drug. NEs usually
exhibit multimodal particle distributions. NCs exhibit monomodal
particle distribution. The manufacture of NEs involves a
multi-component/multi-step process which is difficult to scale up
and detrimental for long-term storage. Further it is difficult to
remove unencapsulated drug and requires difficult to use filtration
method for sterilization. In contrast, the manufacture of NC
involves a single component system which is straightforward to
scale up and when properly stored, drug release should be minimal
increasing storage lifetime. Because there is essentially no free
drug or drug aggregate to remove, the method is simpler and less
costly to implement.
[0054] During polymerization as illustrated in FIG. 2 part A, the
chemical species that functions for polymerization initiation
(e.g., drug) becomes covalently bonded to one or more growing
oligomer or polymer chains. The polymerization reaction is a
ring-opening polymerization reaction which preferably has the
characteristics of a living polymerization. The invention has been
exemplified herein with drugs and other chemical species having one
or more hydroxyl groups or thiol groups which can function in the
presence of certain catalysts as polymerization initiators. As
discussed herein the ring-opening polymerization can employ various
cyclic monomers, including cyclic esters, cyclic carbonates as well
as cyclic siloxanes and cyclic phosphorous containing monomers. The
polymerization can be exemplified for the polymerization of a
lactide or glycolide and with a chemical species which is a drug
and which carries one or more hydroxyl or thiol groups.
[0055] Numerous alcohol-metal oxides (RO-M) have been developed for
controlled, living polymerization of lactide and other related
cyclic monomers with quantitative, terminal conjugation of RO to
polylactide through an ester bond..sup.16 The amount of RO in the
resulting polylactide can be precisely controlled by adjusting
lactide/ROH ratio (e.g., the monomer to initiator molar ratio). In
the present invention, ROH is a drug or other chemical species
containing one or more hydroxyl groups that are to be conjugated to
the polymer formed on ring-opening polymerization. A number of
organocatalysts, such as TBD (1,5,7-Triazabicyclo[4.4.0]dec-5-ene)
can also be employed with hydroxyl or thiol containing initiators
(i.e., drugs or other species to be conjugated) to form the
conjugates of this invention. In these cases as well, the amount of
the drug or other chemical species in the resulting polymer (or
oligomer) is controlled by controlling the monomer/initiator ratio.
Additional exemplary catalysts are provided in Example 6. The
effect of varying catalyst on the actual MW of durg-polymer
conjugates is illustrated in FIG. 12.
[0056] As part of this work, it was demonstrated that
hydroxyl-containing chemotherapeutics could be quantitatively
incorporated into polylactide using this polymerization method
(exemplified with Ptxl in FIG. 2 part B using a Mg(II) complex
((BDI)MgN(TMS).sub.2 to activate Ptxl). As a consequence, drug
release rate from the particle can be modulated by the cleavage of
drug-polylactide ester bond, which is much more controllable than
the diffusion of the encapsulated non-covalently bonded drug from a
particle. Release kinetics of the drug would then be controlled by
adjusting drug loading and particle size. Because polymerization
reactions can be controlled to give quantitative yield, drug
loading can in turn be precisely controlled simply by adjusting
monomer/drug (or other species) molar ratio (monomer to initiator
ratio). The capability of the present methods to precisely control
drug loading by controlling the drug-polymer composition will
significantly enhance clinical translation of the nanoparticle
products and the likelihood for regulatory approval of the
nanoparticles for clinic use. In addition, unprecedented high drug
loading have been demonstrated (up to .about.40%) with
nanoparticles generated by the methods of this invention.
[0057] Nanoparticles can be formed from polymer and/or oligomer
conjugates of this invention by various known methods. In a
specific embodiment, nanoprecipitation is employed in which a
solution of the conjugate is added to a solution in which the
conjugate is insoluble. The precipitation step for forming
nanoparticles employing the conjugates of this invention is
simplified compared to the use of other starting materials because
only one type of material, the conjugate is involved. The
precipitation and encapsulation of a free drug in a polymer, even
in a binary system, in contrast can be very complex. Control of
integration of drug and polymer during phase separation is often
poor especially when these two elements have distinct chemical and
physical properties. Biphasic particle distributions have been
consistently observed in nanoencapsulates which may be due in part
to the self-aggregation of drug or polymer according to the
like-dissolves-like principle. The method of this invention
provides particles with monomodal particle distributions.
[0058] The benefits described above for drug-polymer or
drug-oligomer conjugates will generally be observed in the
formation of conjugates with any chemical species which can
function as polymerization initiators and which it is desired to
delivery in vivo in particulate form. Further, the specific
benefits described above for the preparation of nanoparticle
delivery compositions will generally be observed when the polymer
or oligomer conjugates are employed to make any size particle that
is useful for in vivo delivery.
[0059] Among various NP preparation methods, nanoprecipitation has
been widely used for preparing NPs for use as encapsulated
chemotherapy drugs. In a typical approach, a degradable hydrophobic
polymer, such as polylactide, is mixed with a hydrophobic drug in a
water-miscible solvent (e.g. THF or DMF) and added to excess water.
Diffusion of the organic solvent into water facilitates the
formation of sub-100 nm sized nano-aggregates with randomly mixed
drug and polymer molecules. More generally any of the many methods
that are known in the art for preparing nanoparticles from polymer
or oligomeric materials can be employed with the conjugates of this
invention.
[0060] In one aspect, the invention relates to a nanoconjugation
technique which integrates drug-initiated cyclic ester (or
carbonate) polymerization and nanoprecipitation to prepare
drug-containing nanoparticles with pre-defined drug loading, near
100% encapsulation efficiency, minimized particle heterogeneity and
significantly reduced burst release effect. In applications for
cancer chemotherapy and particularly with nanoparticles useful in
such therapy, particulate formulations of this invention will
exhibit improved efficacy and decreased toxicity.
[0061] In an embodiment the invention relates to polymeric
nanoparticles for cancer treatment. In this aspect of the
invention, the drug-polymer or drug-oligomer conjugate includes an
anticancer agent or chemotherapeutic agent. Nanoparticles useful
for cancer treatment may contain a mixture of conjugates with two
or more anticancer agents.
[0062] Particulate formulations (i.e., those containing NP and NC)
of this invention can be administered to a subject by any known
method appropriate for the size of the particle and the
therapeutic, diagnostic or other agent carried in the
particulate.
[0063] This invention additionally relates to the use of
drug-conjugates with polymers and oligomers in the preparation of a
medicament for in vivo delivery of the drug. The drug can, for
example, be an anticancer agent. More specifically, the invention
relates to the use of a drug in the manufacture of a medicament for
treatment of cancer. In specific embodiments the medicament
manufactured is in the form of particles, particularly
nanoparticles, for administration in any appropriate dosage form.
In specific embodiments, the medicament further comprises a
pharmaceutically acceptable carrier or diluent and particularly a
carrier or diluent suitable for the desired form of
administration.
[0064] The term drug is employed herein very generically to include
any chemical species that can provide therapeutic benefit to an
individual in need of such benefit. Conjugates here can be formed
with appropriate drugs or other chemical species which one wishes
to deliver in a nanoparticle. The drug or other chemical species
must have at least one functional group that can function in the
presence of a catalyst for initiation of ring-opening
polymerization. It is believed that the functional group must be
capable of interaction with the catalyst to form a species active
for polymerization. Hydroxyl groups and thiol groups, for example,
are capable of functioning for initiation of ring-opening
polymerization. Hydroxyl and thiol groups can be primary, secondary
or tertiary functional groups. As is understood in the art,
primary, secondary and tertiary hydroxyl and thiol group have
different steric environments and can exhibit different relative
reactivities.
[0065] In the description of chemical groups herein the terms used
are intended to have their broadest art-recognized meaning.
[0066] The chemical species having at least one functional group
functional for initiation of ring-opening polymerization is
combined with one or more cyclic monomers which can be polymerized
by ring-opening polymerization and an appropriate ring-opening
polymerization catalyst in an appropriate solvent under conditions
and for a sufficient time to form oligomers or polymers as desired.
A variety of chemical species are known to function for initiation
of ring-opening polymerization in the presence of appropriate
catalysts. Among these chemical species are those which contain one
or more hydroxyl groups or one or more thiol groups in their
chemical structure. The ability of a given chemical species to
function for polymerization initiation as required for this
invention can be readily assessed without undue experimentation in
test polymerization reactions carried out employing materials and
methods as taught herein or as well-known in the art. The methods
of the invention have been, for example carried out successfully
with drug and other species illustrated in FIG. 8. FIG. 13 contains
a number of drugs containing hydroxyl groups that are useful in the
preparation of drug-conjugates and nanoconjugate particles of this
invention.
[0067] Additional drugs carrying hydroxyl groups which are useful
in the methods of this invention include, among others, Darunavir
(TMC-114), Tipranavir (TPV), Saquinavir (SQV), Ritonavir (RTV),
Indinavir, Nelfinavir (NFV), Amprenavir (APV), Lopinavir (ABT-378),
Atazanavir (ATV), Vinorelbine bitartrate, fulvestrant,
Sarcodictyins, camptothecins, Vinblastine, bryostatin 1,
(+)-Cylindricine, (+)-Lactacystin, Aeruginosin 298-A,
(+)-Fostriecin, Garsubellin A/Hyperforin, (S)-Oxybutynin,
Epothilone A, Zidovudine (AZT), Lamivudine (3TC), Didanosine (ddI),
Abacavir (ABC), and Emtricitabine (FTC)
[0068] Additional drugs useful in the methods of this invention
include those of various structures, but which have phenolic
hydroxyl groups, which include among others include, bamethane,
ethamivan, hexachlorophene, salicylanilide, pyrocatechin, thymol,
pentazocine, phloroglucinol, eugenol, niclosamide, terbutaline,
dopamine, methyldopa, norepinephrine, eugenol, .alpha.-naphthol,
polybasic phenols, adrenaline, dopamine, phenylephrine,
metaraminol, fenoterol, bithionol, alpha-tocopherol, isoprenaline,
adrenaline, norepiniphrine, salbutamol, fenoterol, bithionol,
chlorogenic acid/esters, captopril, amoxicillin, betaxolol,
masoprocol, genistein, daidzein, daidzin, acetylglycitin, equol,
glycitein, iodoresiniferatoxin, SB202190, and tyrphostin
SU1498.
[0069] For a given chemical species that it is desired to conjugate
by the method herein, it may be necessary to perform trial
polymerizations employing different catalysts, for example, certain
chemical species will be more compatible with organometallic
catalysts, while others may be more compatible with
organocatalysts. For example, it has been found that the chemical
species even though containing appropriate functional groups may
not function (or may have limited function) to initiate
polymerization with certain metal-based catalysts because the
chemical species may deactivate the catalysts. Specifically,
conjugation of PLA with mitoxantrone employing (BDI)MgN(TMS).sub.2
did not proceed. It is believed that the mitoxantrone (Formula J)
which has hydroxyalkyl amine groups may have deactivated the Mg
catalyst.
##STR00001##
[0070] Any of the cyclic monomers described herein including AB2
type cyclic monomers can be employed to form polymer or oligomer
conjugates with such chemical species that can function for
polymerization initiation. Any cyclic monomer or mixtures thereof
that can be polymerized by ring-opening polymerization can be
employed to form the drug-conjugates and particles, particularly
nanoparticles, of this invention. In particular, cyclic monomers
that can be polymerized by activated --OH or a metal-oxide group
can in general be employed to form the drug-conjugates and
particles of this invention. Useful cyclic monomers include cyclic
esters and cyclic carbonates. Cyclic esters include, lactones,
cyclic diesters, and cyclic ester-amides, e.g., cyclic
depsipeptides.
[0071] Cyclic esters have the formula:
##STR00002##
where m+n ranges from 1-20, X is O or NH, x is 0 or 1 to indicate
the presence of the ester or amide group and Y.sub.1 and Y.sub.2
indicate the optional substitution of one or more carbon atoms of
the ring with non-hydrogen substituents. Each Y.sub.1 and Y.sub.2,
independently of one another are substituents that do not interfere
with the polymerization reactions as described herein and can for
example be selected from the group consisting of hydrogen, halogen,
--COOR, --NRR', --SR, --OR, where R and R' independently are one or
more hydrogens, alkyl or aryl groups, a guanidinium group, an
imidiazole group, an alkyl group, alkenyl group, alkynyl group,
aryl group (including phenyl or benzyl) and --N.sub.3. Each Y.sub.1
or Y.sub.2 can also be an amino acid or short peptide having 1-5
amino acids. Each Y.sub.1 or Y.sub.2 also include groups as listed
above which are protected with an art-recognized protecting group.
Alkyl, alkenyl, alkynyl and aryl groups are optionally substituted
with one or more halogens (including one or more fluorines),
--N.sub.3, --COOR'', --NR''R''', --SR'', --OR'' where R'' and R'''
are independently hydrogen or an unsubstituted alkyl, alkenyl,
alkynyl or aryl group. In a specific embodiment, one or two of
Y.sub.1 and Y.sub.2 can be a hydroxyl alkyl group. In specific
embodiments, each Y.sub.1 and Y.sub.2 is a hydrogen or an alkyl
group having from 1 to 6 carbon atoms, particularly a methyl
group.
[0072] Cyclic carbonates have the formula:
##STR00003##
where p ranges from 1-20 and Y.sub.1 and Y.sub.2 indicate the
optional substitution of one or more carbon atoms of the ring with
non-hydrogen substituents and where each Y.sub.1 and Y.sub.2 are as
defined above. In specific embodiments, each Y.sub.1 and Y.sub.2 is
a hydrogen or an alkyl group having from 1 to 6 carbon atoms,
particularly a methyl group. In a specific embodiment, one or two
of Y.sub.1 and Y.sub.2 can be a hydroxyl alkyl group.
[0073] Cyclic esters include, without limitation, lactones such as
.beta.-butyrolactone (n=2), .delta.-valerolactone (n=4),
.epsilon.-caprolactone (n=5), .alpha.-methyl-.beta.-propriolactone,
.beta.-methyl-.beta.-propriolactone, .omega.-pentadecalactone,
.omega.-dodecalactone and any lactide or glycolide including all
stereo-isomers thereof, e.g.
##STR00004##
any substituted lactide or glycolide:
##STR00005##
any cyclic depsipeptides (half-ester and half-amide) with 6 or 7
member ring structure, including, among others, Formulas D1-D3:
##STR00006##
respectively.
[0074] Other cyclic monomers that are polymerizable by activated
--OH or metal-oxide group, include phosphorus-containing cyclic
esters including cyclic phosphates and phosphonates:
##STR00007##
where q=1 to 20, Y.sub.3 is as defined for Y.sub.1 and Y.sub.2
above and R.sub.3 is Y.sub.3 (phosphonates) or --OY.sub.3
(phosphates).
Cyclic Phosphonites:
##STR00008##
[0075] where variables are as defined above, and silicon-containing
cyclic monomers including
##STR00009##
where each R is independently selected from hydrogen or an
optionally substituted alkyl group. In specific embodiments of the
above cyclic monomers, each of Y.sub.1-3 are hydrogen or alkyl
groups having 1-6 carbon atoms. In specific embodiments of the
above cyclic monomers, all Y.sub.1-3 are hydrogen or all Y.sub.1-3
are alkyl groups having 1-6 carbon atoms, particularly all
Y.sub.1-3 are methyl groups. In specific embodiments of the above
cyclic monomers, each R is selected from hydrogen or an alkyl group
having 1-6 carbon atoms. In specific embodiments of the above
cyclic monomers, all R's are hydrogen or all R's are alkyl groups
having 1-6 carbon atoms, particularly all R's are methyl
groups.
[0076] In specific embodiments, AB2 type cyclic polymerizable
monomers are employed alone or in combination with other cyclic
esters or cyclic carbonates. AB2 type cyclic ester monomers include
those of formula:
##STR00010##
where z is 1 to 6 and Y.sub.1 is as defined above. In specific
embodiments, Y.sub.1 can be hydrogen or an alkyl group having from
1-6 carbon atoms.
[0077] The term "alkyl" refers to a monoradical of a branched or
unbranched (straight-chain or linear) saturated hydrocarbon and to
cycloalkyl groups having one or more rings. Unless otherwise
indicated preferred alkyl groups have 1 to 20 carbon atoms and more
preferred are those that contain 1-10 carbon atoms. Short alkyl
groups are those having 1 to 6 carbon atoms including methyl,
ethyl, propyl, butyl, pentyl and hexyl groups, including all
isomers thereof. Long alkyl groups are those having 8-20 carbon
atoms and preferably those having 12-20 carbon atoms as well as
those having 12-20 and those having 16-18 carbon atoms. The term
"cycloalkyl" refers to cyclic alkyl groups having preferably 3 to
20 carbon atoms having a single cyclic ring or multiple condensed
rings. Cycloalkyl groups include, by way of example, single ring
structures such as cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cyclooctyl, and the like, or multiple ring structures
such as adamantanyl, and the like. Unless otherwise indicated alkyl
groups including cycloalkyl groups are optionally substituted as
defined below.
[0078] The term "alkenyl" refers to a monoradical of a branched or
unbranched unsaturated hydrocarbon group having one or more double
bonds and to cycloalkenyl group having one or more rings wherein at
least one ring contains a double bond. Unless otherwise indicated
preferred alkyl groups have 1 to 20 carbon atoms and more preferred
are those that contain 1-10 carbon atoms. Alkenyl groups may
contain one or more double bonds (C.dbd.C) which may be conjugated
or unconjugated. Preferred alkenyl groups are those having 1 or 2
double bonds and include omega-alkenyl groups. Short alkenyl groups
are those having 2 to 6 carbon atoms including ethylene (vinyl),
propylene, butylene, pentylene and hexylene groups including all
isomers thereof. Long alkenyl groups are those having 8-20 carbon
atoms and preferably those having 12-20 carbon atoms as well as
those having 12-20 carbon atoms and those having 16-18 carbon
atoms. The term "cycloalkenyl" refers to cyclic alkenyl groups of
from 3 to 20 carbon atoms having a single cyclic ring or multiple
condensed rings in which at least one ring contains a double bond
(C.dbd.C). Cycloalkenyl groups include, by way of example, single
ring structures (monocyclic) such as cyclopropenyl, cyclobutenyl,
cyclopentenyl, cyclohexenyl, cyclooctenyl, cylcooctadienyl and
cyclooctatrienyl as well as multiple ring structures. Unless
otherwise indicated alkyl groups including cycloalkyl groups are
optionally substituted as defined below.
[0079] The term "alkynyl" refers to a monoradical of an unsaturated
hydrocarbon having one or more triple bonds (C.ident.C). Unless
otherwise indicated preferred alkyl groups have 1 to 20 carbon
atoms and more preferred are those that contain 1-10 carbon atoms.
Alkynyl groups include ethynyl, propargyl, and the like. Short
alkynyl groups are those having 2 to 6 carbon atoms, including all
isomers thereof. Long alkynyl groups are those having 8-20 carbon
atoms and preferably those having 12-20 carbon atoms as well as
those having 12-16 carbon atoms and those having 16-18 carbon
atoms. The term "cycloalkynyl" refers to cyclic alkynyl groups of
from 3 to 20 carbon atoms having a single cyclic ring or multiple
condensed rings in which at least one ring contains a triple bond
(C.ident.C). Unless otherwise indicated alkyl groups including
cycloalkyl groups are optionally substituted as defined below.
[0080] The term "aryl" refers to a monoradical containing at least
one aromatic ring. The radical is formally derived by removing a H
from a ring carbon. Aryl groups contain one or more rings at least
one of which is aromatic. Rings of aryl groups may be linked by a
single bond or a linker group or may be fused. Exemplary aryl
groups include phenyl, biphenyl and naphthyl groups. Aryl groups
include those having from 6 to 30 carbon atoms and those containing
6-12 carbon atoms. Unless otherwise noted aryl groups are
optionally substituted as described herein. The term aryl includes
"arylalkyl" groups which refers to a group that contains at least
one alkyl group and at least one aryl group, the aryl group may be
substituted on the alkyl group (e.g., benzyl,
--CH.sub.2--C.sub.6H.sub.5) or the alkyl group may be substituted
on the aryl group (e.g., tolyl, --C.sub.6--H.sub.4--CH.sub.3).
Unless otherwise noted either the alkyl or the aryl portion of the
arylalkyl group can be substituted as described herein.
[0081] The polymerization reaction to form conjugates of the
invention can be carried out under various reaction conditions
(temperature, solvent, concentrations) as is understood in the art.
These conditions are in part selected to retain activity of any
chemical species, particularly a drug, that is to be conjugated.
The polymerization reaction can be carried out in any appropriate
solvent or mixture of solvents. In a specific embodiment, the
solvent is an anhydrous, water-miscible solvent. The polymerization
can be carried out in the same or a different solvent than that
which is used in the later preparation of particles. Useful
solvents for the polymerization reaction include, among others,
THF, acetone, methylene chloride, chloroform, dimethylformamide,
DMSO, acetonitrile or mixtures thereof.
[0082] The term polydispersity is used herein to refer to the
distribution of molecular weights of polymers in a given sample.
The Polydispersity Index (PDI) is a specific measure of
polydispersity and is the weight average molecular weight divided
by the number average molecular weight and relates to the
distribution of individual molecular weights in a given sample of
polymers. PDI can be determined using Gel Permeation Chromatography
(GPC). As the polymer chains in a given sample approach uniform
chain length, PDI approaches 1.
[0083] Particles of this invention can be surface-modified as is
known in the art to improve their usefulness as drug delivery
vehicles.
[0084] For particles, particularly nanoparticles, that can
successfully carry drug molecules or other chemical species to a
desired in vivo location, e.g., a tumor site and get into cancer
cells, it is preferably to well-control their features so that the
they can circumvent various physiology barriers to reach tumor
tissues. Systemically administered nanoparticles without proper
modification are usually cleared rapidly from the circulation and
localized predominately in liver and spleen. Severe liver and
spleen retention not only greatly diminishes the accessibility of
the nanoparticles to target tissue, e.g., tumor tissue, but also
causes liver and spleen damage. Clearance is due to the scavenging
by liver Kupffer cells and spleen macrophages. Nanoparticles can be
cleared within a few to tens of minutes by this passive and
site-specific mechanism. In addition, nanoparticle surface
characteristics and sizes play an important role in the blood
opsonization, a process of the deposition of opsonins, like
fibronectin, which will trigger immune responses and accelerate the
clearance of nanoparticles from blood by macrophages. The binding
of opsonins to the surface of nanoparticles can be substantially
reduced when surface features of the nanoparticles are well
controlled.
[0085] For example, nanoparticle surface pegylation, a
well-established approach to reduce protein binding, forms a
hydrophilic layer that can substantially reduce blood protein
binding and reduce liver and spleen uptake. Pegylation creates
stealth-like structures resembling the strategies developed by
pathogenic microorganisms to bypass immune detection. Suppression
of opsonization is thus achievable and has been utilized to enhance
passive retention of nanoparticles in circulation and avoid
trapping of nanoparticles in macrophages when they are in contact
with blood. This simple strategy for manipulation of the
nanoparticle surface can have a significant impact, as the
circulation half-life of a nanoparticle can be increased from
several minutes to several or tens of hours on pegylation. The use
of surface pegylation has become a very popular approach to reduce
recognition by macrophage cells.
[0086] Besides surface morphology, nanoparticle size is another
important parameter that can significantly affect the
biodistribution and in vivo efficacy. Nanoparticle sizes can
dramatically affect the clearance rate. Large particles with size
200 nm or above are more likely to induce macrophage immune
response and activate the uptake by Kupffer cells than their
smaller counterparts. The size of fenestrae in the sinus
endothelium in liver can be as large as 150 nm. Splenic filtration
at interendothelial cell slits can predominate when particles size
exceeds that of the cell slits (200-250 nm). Therefore nanoparticle
sizes are usually controlled to 150 nm or below when they are to be
used in anticancer drug delivery in order to have prolonged
circulation. However, the nanoparticle size should not be too
small, otherwise the particles can be very quickly filtered through
the kidney (size <10 nm) which is a typical problem of
polymer-drug conjugates with molecular weight of 40 kDa or lower.
Very small particles (1-20 nm) can also slowly extravasate from the
vasculature into the interstitial spaces, and are further
accumulated in lymph nodes via lymphatic vessels. Nanoparticles
with size smaller than 20 nm can readily escape from the
vasculature into blood capillaries with open fenestration.
Therefore nanoparticles must be large enough to prevent undesirable
leakage from circulation, but must be small enough to minimize
immune responses.
[0087] Most nanoparticles used for anticancer delivery are in the
range of 20 to 150 nm. To improve anticancer delivery, attention
has been focused on the development of stealth technologies to
provide means for increased extravasation of long circulating NPs
at leaky tumor vasculature. The vasculature of tumors is highly
heterogeneous. Depending on the specific location, tumor tissue can
be vascularly necrotic or extremely vascularized so that adequate
nutrient and oxygen can be transported to the tumor tissue to
support its fast growth. Tumor blood vessels are also very
heterogeneous and have several abnormalities when compared to
normal blood vessels. In general, tumor blood vessels are leakier
than their normal counterparts, and are shown to have a
characteristic pore cutoff size ranging between 380 and 780 nm.
These pores become the pathway for NPs to leave the circulation
system and enter the tumor interstitial space. Therefore NP with
size of 150 nm or lower can freely diffuse through these leaky
vessel pores, while particles with sizes larger than 400 nm are
much less likely to extravasate into tumor issue. Because of the
undeveloped lymphatic drainage system in tumor tissue,
nanoparticles which extravasate the leaky pore of tumor vasculature
cannot be readily removed. Therefore sustained circulation results
in increased accumulation of nanoparticles over the time. This
effect is the extremely well-known Enhanced Permeation and
Retention (EPR) effect passive targeting mechanism in caner drug
delivery.
[0088] The polymer and oligomer conjugates of this invention can be
chemically modified by reaction to introduced desired terminal
functional groups. Terminal functional groups of interest for
applications to drug delivery include among others, hydroxyl,
thiol, amine, azide, alkyne, alkene, ketone, phenol, halide,
imidazole, guanidinium, carboxylate, or phosphate groups. These
desired functional groups can be introduced at the terminus of the
polymers or oligomers herein employing well known chemical methods.
These functional groups can be employed to further conjugate the
polymer or oligomer conjugate of this invention with other chemical
species, such as other polymers, other oligomers, carbohydrates,
peptides, proteins, antibodies, nucleic acids, aptamers etc. and/or
to provide sites for surface modification for nanoparticles
prepared using the conjugates of this invention.
[0089] The invention also relates to multiple layer particles in
which a particle prepared by the methods herein is treated to coat
or otherwise provide a second layer of polymer on the nanoparticle.
The second polymer may be the same of different from that of the
polymer of the polymer conjugate in the particle. Particles of this
invention may contain two or more conjugated chemical species,
e.g., two or more different drugs, that are compatible in a given
application. Particles of the invention may contain different
layers or portions in which the concentration of the chemical
species or drug is different. For example an outer layer may
contain a higher or lower concentration of a given chemical species
(e.g., drug) compared to an inner layer. For example, an outer
layer may contain PEG while an inner layer contains a conjugate of
a different polymer. For example, a first inner layer can contain a
polymer or oligomer conjugate of a first drug, and a second outer
layer containing a polymer or oligomer conjugate of a second
drug.
[0090] Nanoparticles of the invention can have a core/shell
structure or have a multiple layer structure in which at least one
of the core or shell or one of the multiple layers is a layer which
is formed from the drug (or other chemical
species)-polymer/oligomer conjugates of this invention. For
example, as illustrated in examples herein the core of a
nanoparticle can be formed form a polymer/oligomer conjugate of
this invention by methods described above for forming
nanoparticles. Thereafter a shell can be added to the core
nanoparticle to generate a core/shell nanoparticle having increased
particle size. More specifically, a core/shell nanoparticle can be
formed with a core that is formed from a first polymer/oligomer
conjugate and a shell that is formed from a polymer, e.g., a
hydrophilic polymer or an amphiphilic polymer. In specific
embodiments the polymer is an amphiphilic block co-polymer. In
specific embodiments, the polymer is a polymer that is a PEG or
which comprises a PEG (as the polymer or as a block of a the
polymer). Alternatively, a core/shell nanoparticle can be formed
from a first polymer/oligomer conjugate of this invention (to form
the core) and a second polymer/oligomer conjugate of this invention
to form the shell. Note that in a specific embodiment, one of the
first or second polymer conjugates can be one in which a label or
reporter molecule is conjugated to the polymer or oligomer. In
specific embodiments, the first and/or second polymer/oligomer
conjugates can be selected from those of a taxane, an anthracycline
antibiotic, or a Shh antagonist which has a functional group, such
as a hydroxyl or thiol group that can function for polymerization
initiation as described herein. In more specific embodiments, the
first and/or second polymer/oligomer conjugates can be selected
from those of Ptxl, Dtxl, Doxo, cyclopamine, or camptothecin.
[0091] Nanoparticles of the invention can be multiple layer
nanoparticles containing three or more different layers wherein at
least one layer is formed from a polymer/oligomer conjugate of this
invention, including those of drugs or other chemical species, such
as labels or reporter molecules. Nanoparticles include those having
three, four or five layers. Nanoparticles include those in which
all layers are formed from polymer/oligomer conjugates of this
invention. Nanoparticles include those in which at least one layer
is formed from a polymer/oligomer conjugate of this invention and
at least one other layer is formed from a polymer (non-conjugated
polymer) such as a hydrophilic, hydrophobic or amphiphilic polymer.
In specific embodiments, nanoparticles include those in which at
least one layer is formed from a polymer/oligomer conjugate of this
invention and at least one other layer is formed from an
amphiphilic polymer comprising PEG.
[0092] Polymers comprising PEG include among others amphiphilic
copolymers comprising PEG such as poly(lactide)-PEG (PLA-PEG) an
amphiphilic copolymer that has a PLA and PEG segment,
poly(glycolide-co-lactide)-b-methoxylated PEG (PLGA-mPEG), an
amphiphilic copolymer that has a PLGA and PEG segment. In such
copolymers, PEG can, for example, range from 10% to 90%, from 20%
to 50%, from 60% to 80%, from 50% to 75%, from 70% to 99% or from
1% to 50% of the copolymer.
[0093] Polymers and oliogmers used in the methods and materials
herein are preferably biocompatible and biodegradable (dependent
upon the desired application). They preferably exhibit little or no
undesired toxicity in use.
[0094] The particles of this invention can be surface-modified for
preferential targeting to certain cell types. Preferential
targeting of particles can for example be achieved by covalent or
non-covalent attachment of targeting ligands to the surface of the
particle.
[0095] The term particle is used herein generally to refer to a
particle having any given shape that has a size that is useful for
in vivo delivery by some administration method. The particles may
be micelles, aggregates, sphere or have no regular shape. The term
particle size is used herein as it is generally used in the art and
is determined by methods described in the Examples herein.
[0096] The invention relates to conjugates of polymers or
oligomers. Most generally a polymer is a chemical species
containing a plurality of repeating units which are bonded to each
other. A polymer may contain more than one different repeating
unit. The repeating unit typically derives from polymerization of a
monomer. A copolymer specifically refers to a polymer containing
two or more structurally different repeating units. The different
repeating units of a polymer may be randomly ordered in the polymer
chain or the same repeating units may be grouped into contiguous
blocks in the polymer. When there are contiguous blocks of the two
or more repeating units in a polymer, the polymer is a block
co-polymer. As used herein the term polymer refers to a chemical
species containing a total of more than 10 repeating units (there
may be one or more repeating units). The term oligomer is used
herein to refer to a chemical species having two to ten repeating
units.
[0097] The conjugates of this invention are formed between a
chemical species which has at least one hydroxyl group or one thiol
group and oligomers or polymers formed by ring-opening
polymerization. The chemical species must contain at least one
functional group which under the conditions of the reaction
functions as an initiator of the polymerization. The hydroxyl group
can most generally be a primary (1'), secondary (2') or tertiary
(3') hydroxyl group attached to a carbon, or a hydroxyl group
attached to carbon of an aromatic ring which is generally described
herein as a "phenolic hydroxyl group." Phenolic hydroxyl groups are
those directly attached to a carbon of an aryl ring. The terms
hydroxyl and hydroxy are used interchangeable herein. Hydroxyl
groups do not include the OH moiety of --COOH groups (carboxylic
acid groups) in which the hydrogen of the group is acidic. The
hydrogens of phenolic hydroxyl groups are more acid than those of
alcohols, but less acidic than those of carboxylic acid groups. The
term hydroxyl as used herein also does not refer to --OH moieties
which are bonded to N, P or S atoms. As is understood in the art a
primary hydroxyl group is a hydroxyl group bonded to a carbon atom
that is also bonded to two hydrogens (e.g., --CH.sub.2--OH). A
secondary hydroxyl group is a hydroxyl group bonded to a carbon
atom that is bonded to one hydrogen atom (e.g. --CH(M)-OH, where M
is an atom or group other than H, in many cases M is a carbon
containing group. A tertiary hydroxyl group is a hydroxyl group
bonded to a carbon atom that is not bonded to a hydrogen, typically
the carbon bonded to the hydroxyl group is bonded to three other
carbon atoms. The thiol group can most generally be a primary (1'),
secondary (2') or tertiary (3') thiol group attached to a carbon,
where the terms primary, secondary and tertiary are used as defined
for the hydroxyl groups.
[0098] The particle formulation of this invention can be used to
treat various diseases, disorders or conditions. Treatment methods
of this invention comprise the step of administering a
therapeutically effective amount of the drug to an individual in
need of treatment in the form of nanoparticles prepared by the
methods of this invention containing the drug. The term
"therapeutically effective amount," as used herein, refers to the
amount a given drug that, when administered to the individual in
the particulate form, is effective to at least partially treat the
disorder, disease or condition from which the individual is
suffering, or to at least partially ameliorate a symptom of such
disorder, disease or condition. As is understood in the art, the
therapeutically effective amount of a given compound will depend at
least in part upon, the mode of administration, any carrier or
vehicle (e.g., solution, emulsion, etc.) employed, the specific
disorder or condition, and the specific individual to whom the
compound is to be administered (age, weight, condition, sex, etc.).
The dosage requirements needed to achieve the "therapeutically
effective amount" vary with the particular compositions employed,
the route of administration, the severity of the symptoms presented
and the particular subject being treated. Based on the results
obtained in standard pharmacological test procedures, projected
daily dosages of active compound can be determined as is understood
in the art.
[0099] Particulate formulations herein can, for example, be in the
form of dry powders which can be rehydrated as appropriate. The
particulate formulations can be in unit dosage forms, e.g. in
capsules, suspensions, dry powders and the like. In such form, the
formulation can be sub-divided in unit dose containing appropriate
quantities of the active ingredient; the unit dosage forms can be
packaged compositions, for example, packaged powders, vials,
ampoules, pre-filled syringes or sachets containing liquids. The
unit dosage form can be, for example, a capsule, or it can be the
appropriate number of any such compositions in package form.
[0100] The dosage employed can vary within wide limits and as is
understood in the art will have to be adjusted to the individual
requirements in each particular case. Any suitable form of
administration can be employed in the method herein. The particles
of this invention can be administered in oral dosage forms,
intravenously, intraperitoneally, subcutaneously, or
intramuscularly, all using dosage forms well known to those of
ordinary skill in the pharmaceutical arts. Compounds of this
invention can also be administered in intranasal form by topical
use of suitable intranasal vehicles. For intranasal or
intrabronchial inhalation or insulation, the compounds of this
invention may be formulated into an aqueous or partially aqueous
solution, which can then be utilized in the form of an aerosol.
[0101] The present invention provides methods of treating
disorders, diseases conditions and symptoms in a mammal and
particularly in a human, by administering to an individual in need
of treatment or prophylaxis, a therapeutically effective amount of
a particulate formulation of this invention to the mammal in need
thereof. The result of treatment can be partially or completely
alleviating, inhibiting, preventing, ameliorating and/or relieving
the disorder, condition or one or more symptoms thereof.
Administration includes any form of administration that is known in
the art to be effective for a given type of disease or disorder,
and is intended to encompass administration in any appropriate
dosage form. An individual in need of treatment or prophylaxis
includes those who have been diagnosed to have a given disorder or
condition and to those who are suspected, for example, as a
consequence of the display of certain symptoms, of having such
disorders or conditions.
[0102] The term drug includes "pharmaceutically acceptable salts"
of drugs as well as prodrugs The term "prodrug," as used herein,
means a compound that is convertible in vivo by metabolic means
(e.g. by hydrolysis) to a drug.
[0103] Particles of the invention can be surface modified by any
known method to improve their surface properties for in vivo
delivery or other applications. Particle surfaces can be modified
for example by pegylation as is known in the art. Particle surfaces
can be modified by coating with a polymer as is known in the
art.
[0104] When a group of substituents is disclosed herein, it is
understood that all individual members of that group and all
subgroups, including any isomers, enantiomers, and diastereomers of
the group members, are disclosed separately. When a Markush group
or other grouping is used herein, all individual members of the
group and all combinations and subcombinations possible of the
group are intended to be individually included in the disclosure. A
number of specific groups of variable definitions have been
described herein. It is intended that all combinations and
subcombinations of the specific groups of variable definitions are
individually included in this disclosure. When a compound is
described herein such that a particular isomer, enantiomer or
diastereomer of the compound is not specified, for example, in a
formula or in a chemical name, that description is intended to
include each isomers and enantiomer of the compound described
individual or in any combination. Additionally, unless otherwise
specified, all isotopic variants of compounds disclosed herein are
intended to be encompassed by the disclosure. For example, it will
be understood that any one or more hydrogens in a molecule
disclosed can be replaced with deuterium or tritium. Isotopic
variants of a molecule are generally useful as standards in assays
for the molecule and in chemical and biological research related to
the molecule or its use. Isotopic variants, including those
carrying radioisotopes, may also be useful in diagnostic assays and
in therapeutics. Methods for making such isotopic variants are
known in the art. Specific names of compounds are intended to be
exemplary, as it is known that one of ordinary skill in the art can
name the same compounds differently.
[0105] Many of the molecules disclosed herein contain one or more
ionizable groups [groups from which a proton can be removed (e.g.,
--COOH) or added (e.g., amines) or which can be quaternized (e.g.,
amines)]. All possible ionic forms of such molecules and salts
thereof are intended to be included individually in the disclosure
herein. With regard to salts of the compounds herein, one of
ordinary skill in the art can select from among a wide variety of
available counterions those that are appropriate for preparation of
salts of this invention for a given application. In specific
applications, the selection of a given anion or cation for
preparation of a salt may result in increased or decreased
solubility of that salt.
[0106] Every formulation or combination of components described or
exemplified herein can be used to practice the invention, unless
otherwise stated.
[0107] Whenever a range is given in the specification, for example,
a temperature range, a time range, or a composition or
concentration range, all intermediate ranges and subranges, as well
as all individual values included in the ranges given are intended
to be included in the disclosure. It will be understood that any
subranges or individual values in a range or subrange that are
included in the description herein can be excluded from the claims
herein.
[0108] All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the invention pertains. References cited herein are
incorporated by reference herein in their entirety to indicate the
state of the art as of their publication or filing date and it is
intended that this information can be employed herein, if needed,
to exclude specific embodiments that are in the prior art. For
example, when composition of matter are claimed, it should be
understood that compounds known and available in the art prior to
Applicant's invention, including compounds for which an enabling
disclosure is provided in the references cited herein, are not
intended to be included in the composition of matter claims herein.
References cited herein are incorporated by reference herein to
provide additional cyclic monomers, additional catalysts,
additional reaction conditions, additional drugs and other chemical
species having at least one hydroxyl group, additional surface
treatment or modification methods and reagents and additional
applications of the methods, compositions and kits of this
invention.
[0109] As used herein, "comprising" is synonymous with "including,"
"containing," or "characterized by," and is inclusive or open-ended
and does not exclude additional, unrecited elements or method
steps. As used herein, "consisting of" excludes any element, step,
or ingredient not specified in the claim element. As used herein,
"consisting essentially of" does not exclude materials or steps
that do not materially affect the basic and novel characteristics
of the claim. In each instance herein any of the terms
"comprising", "consisting essentially of" and "consisting of" may
be replaced with either of the other two terms. The invention
illustratively described herein suitably may be practiced in the
absence of any element or elements, limitation or limitations which
is not specifically disclosed herein.
[0110] One of ordinary skill in the art will appreciate that
starting materials, e.g., cyclic monomers, drugs and other chemical
species having at least one hydroxyl group, biological materials,
reagents, e.g., ring-opening polymerization catalysts, synthetic
methods, purification methods, analytical methods, assay methods,
and biological methods other than those specifically exemplified
can be employed in the practice of the invention without resort to
undue experimentation. All art-known functional equivalents, of any
such materials and methods are intended to be included in this
invention. The terms and expressions which have been employed are
used as terms of description and not of limitation, and there is no
intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed. Thus, it
should be understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this invention as defined by the appended claims.
EXAMPLES
Example 1
Polylactide-Paclitaxel Nanoconjugate Particles
[0111] Nanoparticle design of this invention is based on the use of
drugs as initiators in ring-opening polymerization reactions to
form drug-polymer (and drug-oligomer) conjugates in which the drug
is covalently bonded to the polymer or oligomer. Because the drug
is used as the initiator of polymerization, the efficiency of
conjugation of the drug to the polymer (oligomer) will be very
high, ideally 100%. Additionally, if all of the drug molecules are
efficiently incorporated into a living polymerization (e.g. where
drug molecules function as initiators), the drug loading percentage
can be precisely controlled by adjusting the monomer/initiator
ratio.
[0112] To initially demonstrate this strategy, paclitaxel (Ptxl)
was used in the presence of an appropriate catalyst to initiate a
living polymerization of lactide. Utilization of molecules
containing hydroxyl groups as initiators for the ring-opening
living polymerization of lactide is well established. Paclitaxel,
the best selling chemotherapy drug in the U.S., contains three
hydroxyl groups.
[0113] Metal-oxides (M-ORs) are well-known initiators for living
ring-opening polymerizations of cyclic esters, such as DL-lactide
(LA) used in this study. They can be prepared in situ by mixing a
hydroxyl-containing compound with an active metal complex, such as
a metal-amido compound (B. M. Chamberlain, M. Cheng, D. R. Moore,
T. M. Ovift, E. B. Lobkovsky, G. W. Coates, J. Am. Chem. Soc. 2001,
123, 3229.) The in situ formed M-ORs can initiate controlled,
living polymerization of LA, resulting in quantitative
incorporation of OR to the PLA terminals and 100% monomer
conversions. It was found that Ptxl can be incorporated into
polyesters through metal-Ptxl oxide mediated polymerization of LA.
Drug loadings can thus be precisely controlled by adjusting LA to
Ptxl ratios. The incorporation efficiency of Ptxl to the resulting
PLA should be 100% as the formation of metal-OR is usually
instantaneous and quantitative. After polymerization, Ptxl
molecules are covalently linked to the terminals of PLA through a
hydrolysable ester linker and are subject to sustained release upon
hydrolysis. The Ptxl-PLA conjugates are employed in
nanoprecipitation to generate polymeric NPs, Nanoconjugates (NC)
containing covalently linked Ptxl.
[0114] To ensure a rapid and complete polymerization of LA
(lactide) at room temperature (BDI)MgN(TMS).sub.2 (Chamberlain, et
al. 2001 supra) a very active catalyst for the polymerization of LA
was employed. (See: FIG. 2 for structure of the catalyst.) Ptxl was
mixed with 1 eq. (BDI)MgN(TMS).sub.2 and the polymerization of LA
was completed within minutes at room temperature with nearly
quantitative incorporation of Ptxl into the resulting PLA (Table
1). It is believed that an in situ formed (BDI)Mg-Ptxl complex
(structure uncharacterized; possibly a monomeric Mg-Ptxl oxide)
initiated polymerization.
[0115] The Ptxl incorporated into the polymer conjugate was
released to its original form and other degradation species after
the Ptxl-PLA was treated with 0.1-1 M NaOH, which demonstrated that
Ptxl was conjugated to PLA through a hydrolysable ester bond.
[0116] Nanoprecipitation of the Ptxl-PLA conjugates resulted in
sub-100 nm NPs (Table 1). To be differentiated from NEs, these NPs
derived from nanoprecipitation of Ptxl-PLA conjugates are called
nanoconjugates (NCs). Specific conjugated polymers are named herein
as Drug (or other chemical species)-LA.sub.n, where the drug or
other chemical species is indicated by a shortened form (e.g.,
Ptxl, Dtxl, Doxo, Pyr or Cy5), and PLA is denoted as LA.sub.n where
n is the M/I ratio. In some cases, NC's, i.e., nanoprecipitates
formed from the conjugated polymer are named NC of Drug-LA.sub.n.
The use of these terms is clear from the context of their use.
[0117] NCs with monomodal particle distributions and low
polydispersities were consistently obtained through the
nanoprecipitation of Ptxl-PLA conjugates. Because the multimodal
distribution of NEs is due in part to the aggregation of the
non-encapsulated free drug (J. Cheng, B. A. Teply, I. Sherifi, J.
Sung, G. Luther, F. X. Gu, E. Levy-Nissenbaum, A. F.
Radovic-Moreno, R. Langer, O. C. Farokhzad, Biomaterials 2007, 28,
869), the monomodal distribution observed with NCs is likely
related to the unimolecular structures of Ptxl-PLA conjugates from
which they are made.
[0118] Both the solvent and the concentration of polymer have
dramatic effect on the sizes of NPs prepared by nanoprecipitation.
Solvent that has higher water-miscibility (e.g., DMF) tends to
diffuse into water faster than a solvent with lower
water-miscibility (e.g., THF or acetone) (Cheng et al, 2007,
supra). When a hydrophobic polymer in a highly water-miscible
solvent is added to water, fast nucleation of polymer aggregation
is anticipated. Thus, the increased numbers of particles due to
rapid nucleation lead to reduced particle sizes when the
concentration of polymer remains unchanged in solution. When the
solvent type and the solvent/water ratio are fixed, the particle
sizes usually show a linear correlation with the polymer
concentrations because the number of particles remain roughly
unchanged at that condition.
TABLE-US-00001 TABLE 1 Formation of drug-PLA nanoconjugates (NC)
with high loadings, high incorporation efficiencies, small particle
sizes and low particle distributions Load Lactide.sup.b Particle
Size + Polydispersity + Entry NC M/I.sup.a (wt %) Conver. IE.sup.c
SD(nm).sup.d SD.sup.d,e 1 Pyr-LA.sub.100 100 1.6 >99% >99%
101.0 .+-. 1.4 0.09 .+-. 0.01 2 Pyr-LA.sub.50 50 3.1 >99%
>99% 107.7 .+-. 2.2 0.07 .+-. 0.01 3 Pyr-LA.sub.25 25 6.1
>99% >99% 102.2 .+-. 1.0 0.06 .+-. 0.01 4 Ptxl-LA.sub.100 100
5.6 >99% >99% 95.1 .+-. 2.7 0.04 .+-. 0.01 5 Ptxl-LA.sub.50
50 10.6 >99% >99% 80.6 .+-. 0.2 0.05 .+-. 0.01 6
Ptxl-LA.sub.25 25 19.2 >99% 97% 55.6 .+-. 0.5 0.04 .+-. 0.01 7
Ptxl-LA.sub.15 15 28.3 >99% 95% 85.5 .+-. 1.4 0.09 .+-. 0.03 8
Dtxl-LA.sub.100 100 5.3 >99% >99% 84.7 .+-. 0.5 0.05 .+-.
0.02 9 Dtxl-LA.sub.25 25 8.3 >99% 98% 64.5 .+-. 0.7 0.05 .+-.
0.02 10 Dtxl-LA.sub.10 10 35.9 >99% 95% 77.9 .+-. 1.5 0.06 .+-.
0.02 11 Doxo-LA.sub.100 100 3.6 >99% >99% 96.3 .+-. 0.7 0.082
.+-. 0.011 12 Doxo-LA.sub.50 50 7.0 >99% >99% 101.6 .+-. 0.6
0.075 .+-. 0.011 13 Doxo-LA.sub.20 25 13.1 >99% >98% 90.8
.+-. 0.9 0.088 .+-. 0.010 14 Doxo-LA.sub.10 10 27.4 >97% >94%
125.2 .+-. 2.3 0.110 .+-. 0.014 .sup.aM/I = monomer/initiator
ratio. For all samples, they are first dissolved in DMF and
dropwise added into water under rapid stirring; .sup.bDetermined by
analyzing unreacted lactide using FTIR (1771 cm - 1);
.sup.cIncorporation Efficiency. Based on RP-HPLC analysis of free
molecules. Incorporation efficiency is used instead of
encapsulation efficiency as drug molecules are conjugated to, not
encapsulated in, polylactide; .sup.dDetermined by dynamic light
scattering, SD = standard deviation; .sup.eWhen polydispersity is
measured by the dynamic light scattering machine, the value is a
statistical index to indicate the dispersity of the particle
size.
[0119] Nanoprecipitation of Ptxl-PLA conjugates followed these
trends. At fixed concentration of Ptxl-PLA conjugate, the sizes of
NCs prepared by precipitating a DMF solution of Ptxl-PLA conjugate
are typically 20-30 nm smaller than those prepared with acetone or
THF as solvent. When nanoprecipitation was carried out using DMF as
solvent at a DMF/water ratio of 1/20 (v/v), the size of
Ptxl-LA.sub.200 NCs showed a linear correlation with the
concentration of Ptxl-LA.sub.200 conjugate, and can be precisely
tuned from 60 nm to 100 nm by changing the concentration of
Ptxl-LA.sub.200. Similar linear correlations were observed when
nanoconjugates were formed with other drug-polymer conjugates by
nanoprecipitation.
[0120] Compared with the conventional nanoprecipitates in which
drug loading and encapsulation efficiency can be extremely low in
smaller particles, the nanoconjugates prepared by the method herein
should all contain exactly the same density of drug (e.g., Ptxl) in
polymer matrix because the polymer-conjugate drug loading remains
unchanged during nanoprecipitation.
[0121] Drug burst release causes undesired side-effect and reduced
therapeutic efficacy in nanoencapsulates. Since the Ptxl release
kinetics of Ptxl-PLA NCs is determined by both the hydrolysis of
the Ptxl-PLA ester linker and the drug diffusion, the release
kinetics of Ptxl from NCs should be more controllable with
significantly reduced burst release effect. Well-controlled Ptxl
release was observed in NCs (FIG. 3). Ptxl released from
Ptxl-LA.sub.50 (10.6 wt %) and Ptxl-LA.sub.25 (19.2 wt %) were 7.0%
and 8.7% at Day 1, and 43% and 70.4% at Day 6, respectively. In
comparison, 89% of Ptxl was released within 24 hrs from Ptxl/PLA NE
(FIG. 3). The release of Ptxl from Ptxl-LA.sub.50 NC was slower
than that from Ptxl-LA.sub.25 NC, presumably because of the higher
MW of Ptxl-LA.sub.50 and more compact particle aggregation. In
fact, lower-loading NCs displaying slower drug release were
observed in all drug-PLA NCs that were studied.
[0122] The in vitro toxicities of NCs are determined by the amount
of Ptxl released; they thus show strong correlation with drug
loadings (FIG. 4). The IC.sub.50s of Ptxl-LA.sub.15, Ptxl-LA.sub.25
and Ptxl-LA.sub.50 NCs with similar sizes (.about.100 nm),
determined by MTT assays in PC-3 cells, are 111, 370 and 855 nM,
respectively. The Ptxl-LA.sub.15 NC has nearly identical IC.sub.50
as free Ptxl (87 nM); while the IC.sub.50 of the Ptxl-LA.sub.50 NC
is an order of magnitude higher. As a result, the toxicity of NCs
can be tuned in a wide range simply by controlling NC drug
loading.
[0123] Surface modification of NPs with poly(ethylene glycol) (PEG)
is a widely used approach for prolonged systemic circulation of NPs
and reduced NP aggregation in blood. (P. Caliceti, F. M. Veronese,
Advanced Drug Delivery Reviews 2003, 55, 1261; R. Gref, Y.
Minamitake, M. T. Peracchia, V. Trubetskoy, V. Torchilin, R.
Langer, Science 1994, 263, 1600.)
[0124] A non-covalent approach to pegylate the NC surface was
initially employed to reduce the efforts of removing unreacted
reagents and by-products. For example,
poly(glycolide-co-lactide)-b-methoxylated PEG (PLGA-mPEG), an
amphiphilic copolymer that has a 13 kDa PLGA and a 5 kDa PEG
segment was used to pegylate the NCs. It has been reported by
Pierri et al. (Journal of Biomedical Materials Research Part A;
2005; 639-647) that the micellation of amphiphilic copolymer
PLA-b-PEG can be significantly eliminated when PEG is above 70% or
below 50% of the hydrophobic block.
[0125] It is expected that the PLGA block forms strong interaction
with NCs through hydrophobic interaction to create a stable PEG
shell. Similar approach has been used previously in NP surface
pegylation. (X. H. Gao, Y. Y. Cui, R. M. Levenson, L. W. K. Chung,
S. M. Nie, Nat. Biotechnol. 2004, 22, 969). Sequential addition of
0.4 to 2 equivalent (in mass) of PLGA-mPEG to Ptxl-LA.sub.200
resulted in a linear increase in particle size from 54.5 nm to
100.3 nm (FIG. 5).
[0126] The PLGA-mPEG modified Ptxl-LA.sub.200 NCs showed
significantly enhanced stability in PBS compared to the untreated
NCs or NCs treated only with mPEG (FIG. 6), indicating the
importance of the hydrophobic PLGA segment to the non-covalent
interaction between PLGA-mPEG and NCs. NCs are subject to
instantaneous dilution after intravenous administration, which may
result in dissociation of PLGA-mPEG from NCs. However, sequential
dilution of the PLGA-mPEG treated Ptxl-LA.sub.200 NCs from 1 mg/mL
to 0.01 mg/mL did not show any increase in particle size in PBS.
This study indicates that the PEG shells formed as described above
should remain tightly bound to the NCs in systemic circulation.
[0127] Surface coating of NCs with PEG can lead to formation of
long circulating nanoparticles. The linear increase of nanoparticle
size observed when PLA-PEG was added to the Ptxl-PLA nanoparticles
indicates the formation of a layered structure on the surface of
the nanoparticle because of the hydrophobic interaction of PLA-PEG
and PLA-paclitaxel. PEG corona were formed readily. Nanoparticles
generated using this method are stable in salt solution. The simple
strategy of making salt-stable nanoparticles will make the
nanoparticles readily transferable to systemic study.
[0128] PEG can also be covalently conjugated to the NCs as is known
in the art. (O. C. Farokhzad, J. J. Cheng, B. A. Teply, I. Sherifi,
S. Jon, P. W. Kantoff, J. P. Richie, R. Langer, Pro. Nat'l Acad.
Sci. USA 2006, 103, 6315).
[0129] Ptxl has three hydroxyl groups at its C-2', C-1 and C-7
positions, respectively. Each of these three hydroxyl groups can
potentially initiate LA polymerizations, resulting in Ptxl-PLA
conjugates with 1 to 3 PLA chains attached to Ptxl. To reduce the
heterogeneity of Ptxl-PLA, it is preferred that Ptxl-PLA conjugate
containing a single PLA chain. It has been found that
polymerization initiation can be controlled at a specific hydroxyl
group of the drug (e.g., Ptxl) to make the PLA conjugate containing
a single PLA chain.
[0130] The three hydroxyl groups of Ptxl differ in steric hindrance
in the order of 2'-OH<7-OH<1-OH. The tertiary 1-OH is least
accessible and typically is inactive. (D. Mastropaolo, A. Camerman,
Y. G. Luo, G. D. Brayer, N. Camerman, Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 6920.) The 7-OH, however, could potentially compete with
the 2'-OH, the most accessible and active hydroxyl group of Ptxl,
for complexing with metal catalyst. In view of the difference in
steric hindrance at the different OH groups, it was postulated that
employing a catalyst with bulky groups, e.g., a metal catalyst with
a bulky chelating ligand might differentiate between 2'- and the
7-OH, and thus preferentially or even specifically form Ptxl-metal
complex through the 2'-OH for LA polymerization.
[0131] Attempts using NMR to determine to which hydroxyl group(s)
the PLA chains were attached were unsuccessful because of the
complexity of the material. To determine if PLA was attached to the
2'-OH or the 7-OH, the Ptxl of Ptxl-PLA was reacted with
tetrabutylammonium borohydride (Bu.sub.4NBH.sub.4). This reagent is
reported (N. F. Magri, D. G. I. Kingston, C. Jitrangsri, T.
Piccariello, J. Org. Chem. 1986, 51, 3239) to quantitatively reduce
the 13-ester bond of Ptxl into Baccatin III (BAC) and
(1S,2R)--N-1-(1-phenyl-2,3-dihydroxypropyl)benzamide (PDB). BAC
contains the 7-OH of PTxl, while PDB contains the 2'-OH of
Pxtl.
[0132] Ptxl-LA.sub.5 was prepared using different metal catalysts:
Mg(N(TMS).sub.2).sub.2, a catalyst without a chelating ligand, and
(BDI)MgN(TMS).sub.2 with a chelating ligand and reacted with
Bu.sub.4NBH.sub.4. With Mg(N(TMS).sub.2).sub.2, both PDB-PLA and
BAC-PLA resulted from the reaction indicating that with this
catalyst polymerization initiated at both the 2'-OH and the 7-OH of
Ptxl. When (BDI)MgN(TMS).sub.2 was used, the amount of BAC-PLA
derived was significantly reduced indicating polymerization of LA
was with this catalyst preferentially initiated at the 2'-OH of
Ptxl.
[0133] Although (BDI)MgN(TMS).sub.2 gave significantly improved
site-specific control in the metal/Ptxl initiated polymerization,
the resulting Ptxl-PLAs typically have fairly broad MWD (e.g.,
Ptxl-LA.sub.200 M.sub.w/M.sub.n=1.47). This observation was
attributed to fast propagation relative to initiation for
polymerization initiated by Mg catalysts. (Chamberlain et al., 2001
supra). The catalyst (BDI)ZnN(TMS).sub.2, has a chelating ligand
identical to that of (BDI)MgN(TMS).sub.2, but is less reactive
compared to its Mg analogue even though it gives significantly
improved polymerization of LA. When (BDI)ZnN(TMS).sub.2 was used in
Ptxl-initiated LA polymerization with M/I=200. The resulting
Ptxl-LA.sub.200 had an extremely narrow PDI (M.sub.w/M.sub.n=1.02)
and the MW M.sub.n (obtained)=28,100 kDa; compared to M.sub.n
(expected)=29, 700 kDa). HPLC analysis of Ptxl-LA.sub.5 that was
prepared with (BDI)ZnN(TMS).sub.2 and then treated with
Bu.sub.4NBH.sub.4 as described above demonstrated that initiation
and polymerization was exclusively at the 2'-OH of Ptxl6
[0134] Drug-initiated polymerization method can be applied to the
preparation of NCs of other hydroxyl-containing therapeutic agents,
as well as to drugs containing one or more thiol groups. For
instance, docetaxel (Dtxl)-LA.sub.10 and camptothecin
(CPT)-LA.sub.10 NCs with very high drug loading (35.9 wt % and 19.5
wt %, respectively), more than 95% loading efficiencies and sub-100
nm sizes are readily prepared using this metal/drug complex
initiated LA polymerization followed by nanoprecipitation (Table
1). CPT differs from both Ptxl and Dtxl as it has no intrinsic
ester bond. It thus was quantitatively recovered from CPT-PLA NC
after being treated with NaOH. The CPT separated from the
hydrolysis mixture of CPT-PLA in PBS and collected on a preparative
HPLC showed a 1H NMR spectrum identical to that of the authentic
CPT. This study further demonstrated that the chemical structures
of the incorporated drugs remain unchanged under the mild
polymerization and nanoprecipitation processes. The incorporated
drugs in NCs can be released to their original forms.
[0135] Like Ptxl-LA NCs, Dtxl-LA NCs, Doxo-LA and CPT-PLA NCs
showed no burst release effects in PBS. The toxicity-drug loading
correlations of both Dtxl-PLA and CPT-PLA NCs are similar to that
of Ptxl-PLA NCs (FIGS. 7A-D).
[0136] The method of this invention allows preparation of
polymer-drug conjugated NPs (NCs) that have very high drug loadings
(up to 35%), nearly quantitative loading efficiencies, controlled
release profiles without burst release effects, and narrow particle
distributions. Safe, nutrient metals are involved in this
formation; and organic chelating ligand can be readily removed via
solvent-extraction. Additionally it takes only a few hours to
prepare gram-scale, high-loading, salt-stable NCs. Drug release
profiles can potentially be further controlled through the use of
different cyclic ester monomers, as discussed above, for preparing
NCs.
[0137] Preparation of Ptxl-PLA NC: (BDI)MgN(SiMe.sub.3).sub.2 (6.2
mg, 0.01 mmol) and Ptxl (8.5 mg, 0.01 mmol) were mixed in 0.5 mL
anhydrous THF. DL-Lactide (144 mg, 1 mmol) in 2 mL anhydrous THF
was added dropwise. After LA was completely consumed (monitored by
FT-IR or 1H NMR), the polymerization solution was precipitated in
ethyl ether (25 mL) to give Ptxl-LA.sub.100 conjugate.
Polymerization using (BDI)ZnN(SiMe.sub.3).sub.2 or
Mg(N(SiMe.sub.3).sub.2).sub.2 were similarly performed. An acetone
or DMF solution of Ptxl-LA.sub.100 (100 .mu.L, 10 mg/mL) was
precipitated dropwise to vigorously stirred nanopure water (2 mL).
PLGA-mPEG5k (MW=18,300 g/mol, 5 mg/mL in DMF, 100 .mu.L) or mPEG5k
(5 mg/mL in DMF, 100 .mu.L) was dropwise added to NCs.
[0138] Characterization and Evaluation of Ptxl-PLA NC: NC sizes
were characterized by a ZetaPALS dynamic light scattering detector
(Brookhaven Instruments, Holtsville, N.Y., USA) or by SEM. The
resulting NCs were purified by ultrafiltration; their in vitro
toxicities were then evaluated using MTT assay in PC-3 cells (24 hr
incubation at 37 C). To determine the release kinetics of Ptxl-PLA,
a PBS solution of NCs was equally divided into several portions and
then incubated at 37 C. At scheduled time, the release study was
terminated. DMF solution was added to dissolve all precipitation.
All the samples were then dried, re-dissolved in DCM and reacted
with Bu.sub.4NBH.sub.4 for 1.5 hours. A drop of acetic acid was
added into the solution. The solution was stirred for 20 min before
the solvent was evaporated. The residual solid was reconstituted in
acetonitrile for RP HPLC analysis (Curosil, 250.times.4.6 mm,
5.mu.; Phenomenex, Torrence, Calif., USA).
[0139] We have demonstrated that conjugate formation using the
strategy illustrated in FIG. 2 for several different small
molecules and for a peptide. The structures of these molecules are
shown in FIG. 8. Representative nanoconjugate data are shown in
Table 1. Exemplary detailed PLA-drug polymerization conditions are
provided below. Nanoparticle formation, characterization, and
release evaluation were carried out similarly to those methods used
for PLA-paclitaxel nanoparticles.
Example 2
Polylactide-Doxorubicin Polymer (Doxo-PLA)
[0140] The catalyst (BDI)MgN(TMS).sub.2 and D,L-lactide were
treated as in Ptxl-PLA polymerization. The polymerization was
conducted in a glove box. All the reaction vessels were covered
with aluminum foil and the box light was turned off. First,
doxorubicin was dissolved in DMF and stirred for 10 min, until it
completely dissolved. (BDI)MgN(SiMe.sub.3).sub.2 was then added to
and dissolved in THF. The doxorubicin and
(BDI)MgN(SiMe.sub.3).sub.2 solutions were mixed for 15-20 min, and
the solution changed color from orange red to purple. On HPLC
analysis, the peak associated with doxorubicin shifted, indicating
that a complex of (BDI)MgN(SiMe.sub.3).sub.2 and doxorubicin was
formed. The UV detector was at 450 nm. D,L-lactide was dissolved in
THF and added dropwise into the mixture of doxorubicin and
(BDI)MgN(SiMe.sub.3).sub.2 with rapid stirring. The reaction
process was monitored by HPLC until all of the doxorubicin was
gone. The UV spectrum of Doxo-LA exhibited an absorption at 325-400
nm, different from the absorption of doxorubicin at 400-500 nm.
Nanoparticles were formed by nanoprecipitation similarly to
Ptxl-PLA nanoparticles.
Example 3
Dtxl-PLA using 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) or
BDI--Mg--N(TMS).sub.2
[0141] The TBD or BDI--Mg--N(TMS).sub.2 catalyst was mixed with
docetaxel and the lactide was added to the mixture of catalyst and
initiator. For example, docetaxel and TBD were dissolved in THF
solution and stirred for 5-10 min. (In HPLC, the peak of docetaxel
shifted, indicating the TBD formed complex with docetaxel).
D,L-Lactide was dissolved in THF solution and added dropwise into
the mixture of docetaxel and TBD. The reaction was similar to that
of paclitaxel-PLA, monitored by FTIR and HPLC. Polymerization
initiated by docetaxel-Mg(II) complex was carried out in the same
way as described above for paclitaxel. Nanoparticles were formed
similarly to Ptxl-LA nanoparticles.
Example 4
PLA-Pyrenemethoxy Polymerization
[0142] Pyrenemethanol (TCI America) was purified by dissolving in
THF with CaH.sub.2 and stirring overnight, filtered and vacuum
dried and stored in glove box freezer. The pyrenemethanol was mixed
with the LA in BDI--Mg--N(TMS).sub.2. The ratio of monomer to
initiator (Pyr) was selected. After 24 hours the reaction was
stopped and purified by washing with methanol and ether for three
times. NMR confirmed the PLA copolymer formation conjugated with
pyrenemethanol. Nanoparticles were formed similarly as Ptxl-PLA
nanoparticles.
Examples 5
PLA-LHRH Polymerization
[0143] Goserelin was obtained from Bachem and stored in a freezer.
(BDI)MgN(SiMe.sub.3).sub.2 and D,L-Lactide were treated as
described above for Ptxl-PLA polymerization. The polymerization was
conducted in a glove box. Goserelin was dissolved in DMF solution
with stirring for 10 min. (BDI)MgN(SiMe.sub.3).sub.2 was dissolved
in THF solution. The goserelin and (BDI)MgN(SiMe.sub.3).sub.2
solutions were mixed for 30 min with stirring. D,L-Lactide was
dissolved in THF and added into the mixture. The ratio of lactide
to goserelin was selected. During polymerization, the solution may
become cloudy indicating precipitation of some species. If this
occurs, more DMSO can be added to keep the components and products
in solution. The conversion of goserelin was detected by HPLC. The
conversion observed was however less than 95%. Nanoparticles were
formed from the groserelin-conjugates as described for Ptxl-PLA
nanoparticles.
Example 6
Zn, Ca and Fe Catalysts, and Organocatalysts
[0144] Mg(II) complexes gave fast polymerization. However, in some
instances Mg(II) may give significantly faster propagation than
initiation which is undesirable for carrying out a living
polymerization. Coates has demonstrated that certain Zn catalysts
facilitate fast initiation and relatively slow chain propagation
and Zn-mediated lactide polymerization can result in polymers with
narrow polydispersity..sup.19 Therefore, (BDI)Zn-(docetaxel) and
(BDI)Zn-(doxorubicin) are useful as initiators in the methods
herein. Other catalysts useful for these methods include those of
Ca and Fe..sup.16 Mg, Zn, Ca, and Fe are elements found in human
body, therefore they have should better safety profile than other
active catalysts such as Al and Sn. Exemplary catalyst include,
among others,
TABLE-US-00002 ##STR00011## 10 ##STR00012## 12 ##STR00013## 13 Ar =
2,6-(i-Pr).sub.2C.sub.6H.sub.3 ##STR00014## 22a M = Zn, X =
OSiPh.sub.3 22b M = Mg, X = OPh ##STR00015## 23 ##STR00016##
##STR00017## 21 21' M = Mg, Ca, Zn X = OEt, OPh,
O[2,6-(i-Pr).sub.2C.sub.6H.sub.3], OSiMe.sub.3 N(SiMe.sub.3).sub.2
R = t-Bu, i-Pr ##STR00018## ##STR00019## complex R.sup.1 R.sup.2
48a Me H 48b Me Me 48c Me t-Bu 48d Me Cl 48e CH.sub.2Ph H 48f
CH.sub.2Ph Me 48g CH.sub.2Ph t-Bu 48h CH.sub.2Ph Cl
L.sub.1ZnN(SiMe.sub.3).sub.2 (29a) L.sub.1ZnN(i-Pr).sub.2 (29b)
(L.sub.1ZnOi-Pr).sub.2 (29c) L.sub.1Zn(OSiPh.sub.2)(THF) (29d)
L.sub.1ZnOt-Bu (29e) L.sub.1ZnOCHMeCO.sub.2Me (29f)
(L.sub.1ZnOAc).sub.2 (29g) L.sub.1ZnEt (29h) L.sub.1SnOi-Pr (30)
L.sub.1Mg[N(i-Pr).sub.2](THF) (31a) (L.sub.1MgOi-Pr).sub.2 (31b)
L.sub.1Mg(Ot-Bu)(THF) (31c) L.sub.1Ca[N(SiMe.sub.2).sub.2](THF)
(32) L.sub.2FeOt-Bu (33), where ##STR00020## ##STR00021## Ar =
2,6-(i-Pr).sub.2C.sub.6H.sub.3 or Ar =
2,6-(Ethyl).sub.2C.sub.6H.sub.3
Example 7
Release Studies
Pyrenemethanol-PLA Release Study
[0145] Free pyrenemethanol was used to calibrate the
concentration-HPLC peak area (or intensity) curve. The nanoparticle
was formed in acetonitrile (10 mg/ml)-water (1/10) system and
washed by DI water three times to remove non-covalently bound small
molecules. At day 0, the PBS 1X-NP solutions were prepared in
several vials and incubate at 37 C. Two vials were used in an
experiment. The content of one vial was centrifuged at 4000 rpm for
30 min to spin down all the NPs and the concentration of
pyrenemethanol concentration in the supernatant was measured. 1N
NaOH solution was added to the other vial at 37 C for 30-90 min to
degrade all the NPs and the total concentration of pyrenemethanol
(100%) was measured. (Before injection into the HPLC, the pH value
of the solution was adjusted to 7 using acetic acid). At a selected
time point, a vial is taken from the 37 C incubator, NPs are spun
down to get the supernatant and prepare to 1/1 PBS-acetonitrile
solution and injected into HPLC. The integrated peak and intensity
was documented and compared with measurement of total 100%
pyrenemethanol concentration in the nanoparticles to determine the
release profile. The HPLC detector monitored for absorption at 227
nm and 265 nm. The mobile phase used is 50/50 acetonitrile/DI water
with 0.05% TFA.
Docetaxel-PLA Release Study
[0146] Free Docetaxel was used to calibrate a standard curve in
HPLC analysis. The PLA-docetaxel nanoparticles were well dispersed
in 1.times.PBS solution and extracted with 1-octanol. The 1-octanol
extract was directly injected into the HPLC. The analysis condition
used were the same as used for PLA-pyrenemethanol, and the detector
monitored at 227 nm and 265 nm. The percentage of release is
determined by the comparison of the amount of docetaxel in 100%
incorporation polymer at the same HPLC injection concentration.
Example 8
Dendritic Nanoconjugates in 2-20 nm Range
[0147] Because drug release kinetics are directly correlated to
nanoparticle surface area, further miniaturization of nanoparticles
will result in even faster drug lease. In addition, small
nanoparticles can have dramatically different properties for drug
delivery and other applications compared to larger particles. For
example, small nanoparticles should have different cell uptake
characteristics. For relatively large particle size (.about.100
nm), particles can be endocytosed. But when the NP is in .about.10
nm range, direct particle penetration or entry through solution
pinocytosis (cell drinking) is possible. Micellation of block
copolymers typically results in particles larger than the 10 nm
range.
[0148] To prepare smaller size nanoparticles, dendritic polymer or
oligomer conjugates can be used. Such conjugates are formed by
polymerization AB2 type cyclic monomers, such as hydroxyl
lactides:
##STR00022##
where z is 1-6 and Y.sub.1 is as defined above. In specific
embodiments Y.sub.1 is H.
[0149] For example, the AB.sub.2 monomer of Formula H where z is 1
and Y1 is H can be used. This monomer is synthesized as illustrated
in Scheme 1 below. Polymerization of the hydroxyl lactide molecule
in the presence of a drug having at least one hydroxyl or thiol
group results in formation of a conjugate having a dendritic or
hyperbranched structure as illustrated in FIG. 9.
[0150] In another specific embodiment, in which the drug or other
chemical species has one hydroxyl group, a single attached polymer
can form a hyperbranched structure around the drug molecule. In a
specific embodiment, in which the drug or other chemical species
has two or more hydroxyl groups, multiple attached polymers can
form a hyperbranched structure around the drug molecule. This
embodiment is exemplified in FIG. 9. It will be appreciated, that
not all hydroxyl groups in the drug or other species, will have the
same reactivity for initiation of polymerization and attachment of
the polymer or oligomer. Thus, for a given drug or other chemical
species, the polymer or oligomer may be formed selectively at fewer
than all hydroxyl groups. Also a given population of
polymer/oligomer conjugates may be heterogeneous with respect to
the number of polymers/oligomers attached to each drug or other
chemical species, i.e., it may be that not all of the conjugates
will have the same number of attached polymer/oligomers. The
formation of sufficient hyperbranched structure results in the
formation of a unimolecular dendritic nanoparticle. This method
employing a cyclic AB2 type monomer can result in the formation of
small particle size nanoparticles (20 nm or less) containing a
selected drug. The resulting nanoparticles are water soluble as the
hyperbranched polyester structure has many peripheral hydroxyl
groups.
##STR00023##
[0151] In the synthesis of Scheme 1, H-Ser(Z)-OH (30.0 mg, 154
mmol) was dissolved in 1M sulfuric acid 400 ml and acetonitrile 400
ml. NaNO.sub.2 (21.7 g 313 mmol) was dissolved in 150 ml water and
added dropwise into the reaction flask over 30 min. The reaction
was stirred for 18-24 hours under nitrogen protection. The solution
was extracted with dichloromethane and ethyl acetate 500 ml (3
times). The combined organic layers were dried with magnesium
sulfate, filtered, and concentrated in vacuo. The resulting product
(25.4 g, 129 mmol) was dissolved in dichlormethane (300 ml) with
17.9 ml triethylamine and added dropwise (over 30 minutes) into
2-bromopropionyl bromide (13.5 ml 129 mmol) and DMAP (1.58 g, 129
mmol) in 150 ml dichloromethane solution in an ice cold bath. The
reaction was thereafter stirred for 18-24 hours under nitrogen. The
mixture was precipitated using ether and the remaining solution was
filtered. The combined organic solution was evaporated to give a
light yellow oil (product of second step)
[0152] Sodium iodine (18.59 g, 1.24 mol, 10 equivalents) was added
into 500 ml acetone solution containing the product of the second
step (4.1 .mu.g, 12.4 mmol) and the mixture was refluxed overnight.
Temperature was controlled to 55-60 C under nitrogen. The reaction
was stopped and cooled down the next day. Solvent (acetone) was
filtered using celite and the acetone was evaporated. The resulting
brown oil was redissolved in acetone and extracted with
Na.sub.2S.sub.2O.sub.3 2M (300 ml)(3 times) to give a yellow
solution which was dried with MgSO.sub.4. Finally the solvent was
evaporated to give a crude product (of step three) which was used
without further purification.
[0153] A solution of this product (4.53 g 12.1 mmol) in 100 ml DCM
was added dropwise to refluxing DIEA (4.6 ml, 27.7 mmol) in 1000 ml
acetone over 8 hours. The temperature was controlled at 70 C. HPLC
confirmed that the reaction was complete. The diastereomers were
separated on a silica gel column by eluting with chloroform and
methyl tert butyl ether (1/1). The solvent was evaporated and the
solid was redissolved in methyl tert butyl ether, and then excess
hexane was added to the solution to precipitate out a white
solid.
[0154] The white solid (1.5 mmol, 375.0 mg) was dissolved in
methanol (1.5 m) and the Cbz group was deprotected by H.sub.2 with
10% Pd/C catalyst (40 mg). After two days the solution was
evaporated to give a white solid. NMR and EI-MS confirmed the
structure.
[0155] The polymerization of the hydroxyl substituted cyclic
monomer (1.0 mmol, 160 mg) is conducted in a glove box. The monomer
is dissolved in 500 ul DMF solution. (BDI)MgN(SiMe.sub.3).sub.2
(0.03 mmol, 18.0 mg) is dissolved in 1 ml THF and mixed with
docetaxel (0.01 mmol, 8.1 mg) in THF solution for 10-30 min. The
monomer solution is very slowly added into the mixture over 10 min.
The reaction is monitored by HPLC at 227 nm and 265 nm. After
finishing the reaction, the polymer is purified by reverse-phase
semi-preparative HPLC using a SEC column. The polymer forms
micelles in water solution and the size of the particles formed is
in the range from 5-30 nm, as characterized by AFM and dynamic
light scattering measurement.
Example 9
Design of Nanoconjugates in 20-60 nm Range
[0156] It is a challenge to make nanoparticles smaller than 60 nm,
particularly by precipitating hydrophobic PLA or Dtxl-PLA.
Micellation can, however, be used to make nanoparticles with
precisely controlled sizes in a range of 20-60 nm. PEG is
conjugated to the terminal --OH group of Dtxl-LA. The conjugation
of PEG can be performed after the polymerization conjugation.
Alternatively, a single reaction step can be employed. After each
polymerization, the terminal hydroxyl group is reactive to capping
groups like isocyanate. PEG-isocyanate, for example, is used as a
capping agent to cap the terminal hydroxyl groups of drug-PLA
conjugate. PEG-isocyanate is readily prepared by reacting
PEG-NH.sub.2 with triphosgene. Reaction of PEG-isocyanate with
Dtxl-LA-OH is fast, and results in Dtxl-LA-urethane linker-PEG in
quantitative yield. After purifying the conjugated polymer through
an SEC column, the purified conjugated polymer is used to formulate
micelles. When PEG with higher average MW is used, micelles with
small particle size (20 nm-60 nm) can be readily generated. The use
of PEG-poly(aspartic acid)-drug conjugates to form copolymer
micelles has been extensively studied by Kataoka,.sup.20
Kwon.sup.21, 22 and the inventors..sup.23, 24
[0157] In a specific example, mPEG5k-NH.sub.2 500 mg (0.1 mmol) was
dissolved in dichloromethane (10 ml) with stirring. Triphosgene
269.8 mg (1 mmol) was added to the solution and the reaction
mixture was refluxed at 50-60 C for at least 2 hours. The reaction
was monitored by FTIR, where the isocyanate peak appears at 2250
cm.sup.-1. The yield is calculated by the titration of the
mPEG5k-NCO with the quantified pyrenemethanol to determine the
concentration of the mPEG5k-NCO in the solution. After the reaction
is complete, the solvent is evaporated and the polymer is washed
with hexane and cold ether (3 times). Unreacted mPEG5k-NH.sub.2 is
separated from the product by a size exclusion column using THF or
acetonitrile/hexane as eluent. The product is dried in vacuo and
stored under a nitrogen atmosphere in the freezer.
Example 10
Double Emulsion Technique Design of Nanoparticles (100-600 nm),
Exemplified with Dtxl-LA Conjugates
[0158] Drug encapsulated microparticles are prepared using a
water-in-oil-in-water (W/O/W) solvent evaporation procedure. In
brief, 50 microliter of water was emulsified in a 1 mL solution of
the polymer conjugate (Dtxl-LA) (50 mg) in dichloromethane using a
probe sonicator (Sonic & Materials Inc, Danbury, Conn., USA) at
10 W for 15-30 s. The emulsion was then poured into 50 mL of
aqueous PVA (1%) or sodium cholate solution (1% w/v) and the
mixture was homogenized for 1 min (8000 rpm). The resulting
emulsion was poured into 150 mL of aqueous PVA or sodium cholate
solution (0.3% w/v) with gentle stirring, after which organic
solvent was evaporated by stirring at RT for 2 h or rapidly removed
using a rotary evaporator. Finally, the nanoparticles were isolated
by centrifugation at 6000 rpm for 30 min, washed with distilled
water, and preserved at -15 C in an emulsion form in distilled
water (6 mL). Alternatively, the nanoparticles can be lyophilized
to obtain a powder.
Example 11
Double Emulsion Method for Preparation of Microparticles (600
nm-100 .mu.m)
[0159] Drug encapsulated microparticles are prepared using the
water-in-oil-in-water (W/O/W) solvent evaporation procedure (double
emulsion method) employed elsewhere. In brief, 50 microL of water
is emulsified in a 1 mL solution of the polymer conjugate (Doxo-LA)
(50 mg) in dichloromethane using a probe sonicator (Sonic &
Materials Inc, Danbury, Conn., USA) at 10 W for 15-30 s. The
emulsion is then poured into 50 mL of aqueous PVA (1%) or sodium
cholate solution (1% w/v) and the mixture is homogenized for 1 min
at a speed of 500-8000 rpm. The resulting emulsion is poured into
150 mL of aqueous PVA or sodium cholate solution (0.3% w/v) with
gentle stirring, after which organic solvent is evaporated by
stirring at room temperature for 2 h or is rapidly removed using a
rotary evaporator. Finally, the nanoparticles are isolated by
centrifugation at 6000 rpm for 30 min, washed with distilled water,
and preserved at -15 C in emulsion form in distilled water (6 mL)
or are lyophilized to obtain a powder.
Example 12
Surface Functionalization Using Herceptin
[0160] Trastuzumab (Herceptin) is dissolved at 1 mg/ml in phosphate
buffer (pH=8.0). 2-iminothiolate (5.7 mg) is dissolved in 5 ml
phosphate buffer at pH 8.0. The 2-iminothiolate solution (8.04
microL) is mixed with 1 ml Trastuzumab solution for 6 hours at 20
C. The resulting thiolated antibody can be purified using a Dextran
Desalting SEC column with phosphate buffer as eluent and detecting
at 280 nm. The antibody solution is further concentrated using a
Microcon 30000 microconcentrator. The thiol group concentration in
the antibody solution can be determined using Ellman reagent. The
antibody solution 250 ul is mixed at room temperature with 6.25 ul
4 mg/ml Ellman reagent (in phosphate buffer pH=8.0) for 15 min, and
detected by the UV spectrometer at 412 nm. The number of thiol
groups is calculated relative to a L-cysteine standard
solution.
[0161] MAL-PEG5k-isocyanate (where, MAL is a malimide group) is
conjugated to Doxo-PLA at room temperature for 8 hours and the
polymer is purified on an SEC column. The polymer is dissolved in
acetone at 10 mg/ml concentration and added dropwise under vigorous
stirring into water with the volume ratio of acetone:water=1/20.
The coupling reaction of thiol of the antibody and the malimide of
the polymer is then conducted in nanopure water solution at room
temperature. The reaction is allowed to continue for at least 12
hours. The SEC column is used to detect unbounded thiolated
antibody in the water phase. After conjugation, the efficiency is
determined by compared the peak area of thiolated antibody from the
SEC column before reaction at the same concentration.
[0162] There is an alternative method for incorporation of
thiolated antibody. The PLGA-PEG-MAL (poly(lactic-glycolic
acid)-poly(ethylene glycol) copolymer with a terminal malimide
group) or PLA-PEG-MAL (poly(lactic acid)-poly(ethylene glycol)
copolymer with a terminal malimide group) can either be purchased
or made by known methods. First the Doxo-LA is dissolved in acetone
and the material is nanoprecipitated to form nanoparticles. Then an
acetone solution of PLGA-PEG-MAL or PLA-PEG-MAL is added dropwise
to the nanoparticle. Use of this method keeps the particle
distribution unchanged. The further conjugation between the
malamide and thiol group of the antibody is performed as described
above.
[0163] After conjugation, the nanoparticle is surface-modified with
the herceptin. The function of the antibody can be tested. The
nanoparticles are incubated with MCF-7 and SK-BR-3 cells for 3 days
after which western blotting analysis is performed. The SK-BR-3
cell that express HER 2 and MCF 7 cells are used as a negative
control.
Example 13
Preparation of Cyclopamine (Ca) Ncs
[0164] The preparation of CA-LA.sub.100 nanoconjugates involved two
steps. The first step was to conjugate CA with PLA polymer.
Briefly, in the glove box, cyclopamine (4.11 mg, 0.01 mmol) was
dissolved in 1.0 mL THF solution. (BDI)MgN(SiMe.sub.3).sub.2 (6.0
mg, 0.01 mmol) was mixed with CA for 5-15 min. DL-Lactide (144 mg,
1.0 mmol) in 1 mL THF solution was added dropwise to the mixture of
CA and (BDI)MgN(SiMe.sub.3).sub.2 with vigorous stirring. FTIR was
used to calculate the conversion of Lactide. The reaction finished
overnight and CA-LA.sub.100 conjugated polymer was obtained. The
second step was to use the polymer solution to directly prepare NPs
by the nanoprecipitation method. CA-LA.sub.100 polymer in DMF
solution was added dropwise into 20.times. nanopure water, a
non-solvent. The resulting NPs suspension can be purified by
ultrafiltration (15 min, 3000 g, Amicon Ultra, Ultracel membrane
with 10,000 NMWL, Millipore, Billerica, Mass.).
Example 14
Preparation of Cyclosporin (Cp) Ncs
[0165] The preparation of CP-LA.sub.100 nanoconjugates involved two
steps. The first step was to conjugate CP with PLA polymer.
Briefly, in the glove box, cyclosporine 12.02 mg (0.01 mmol) was
dissolved in 1.0 mL THF solution. (BDI)MgN(SiMe.sub.3).sub.2 (6.0
mg, 0.01 mmol) was mixed with CA for 5-15 min. DL-Lactide (144 mg,
1.0 mmol) in 1 mL THF solution was added dropwise to the mixture of
CA and (BDI)MgN(SiMe.sub.3).sub.2 with vigorous stirring. FTIR was
used to calculate the conversion of Lactide. Finally the reaction
finished overnight and CA-100 polymer was obtained. The second step
was to use the polymer solution to directly prepare NPs by
nanoprecipitation method. In general, CA-100 polymer in DMF
solution was dropwise added into 20.times.nanopure water, a
non-solvent. The resulting NPs suspension can be purified by
ultrafiltration (15 min, 3000 g, Amicon Ultra, Ultracel membrane
with 10,000 NMWL, Millipore, Billerica, Mass.).
Example 15
Preparation of Core-Shell Nanoconjugates (50-200 nm Range)
Including Those Containing Two or More Drugs
[0166] Doxo-LA.sub.25 polymer (5 mg/mL in DMF, 100 .mu.L) was added
dropwise to 2 mL nanopure water to give Doxo-LA.sub.25 NCs.
PLGA-mPEG5k (MW=18,300 g/mol, 5 mg/mL in DMF, 100 .mu.L) or mPEG5k
(5 mg/mL in DMF, 100 .mu.L) was then added dropwise to Doxo-25 NP.
The NC that resulted are core-shell NCs where the core is Doxo-LA25
and the shell is PLGA-mPEG5k or mPEG5k.
[0167] Ptxl-LA.sub.200 conjugate (2 mg/mL in DMF, 100 .mu.L) was
added dropwise to 2 mL nanopure water to give Ptxl-LA.sub.200 NCs.
PLA-PEG3k (MW=17,500 g/mol, 2 mg/mL in DMF) was then sequentially
added into Ptxl-LA200 NCs solution under vigorous stirring. The
resulting NCs are core-shell NCs.
[0168] Analogous methods can be employed with drug-polymer
conjugate prepared by the methods herein to form analogous
nanoconjugates with core-shell structure.
[0169] Dtxl-LA.sub.100(core)/Doxo-LA.sub.100 (Shell) NCs:
Dtxl-LA.sub.100 in DMF (10 mg/mL, 100 .mu.L) was added dropwise
into 2 mL nanopure water to form nanoparticles (NCs) and thereafter
Doxo-LA.sub.100 in DMF (10 mg/mL, 100 .mu.L) was further added into
the nanoparticle solution with vigorous stirring (3000 rpm) at 50
.mu.L/min rate. The resulting NCs are dual drug core-shell NCs
illustrated in FIG. 10A. In this figure the shift in particle size
from about 80 to about 100 nm is illustrated.
[0170] Ptxl-LA.sub.100/CPT-LA.sub.100: Ptxl-100 in DMF (10 mg/mL,
100 .mu.L) was added dropwise into 2 mL nanopure water and the
CPT-100 in DMF (10 mg/mL, 100 .mu.L) was further added into the
nanoparticle solution with vigorous stirring (3000 rpm) at 50
.mu.L/min rate.
[0171] These methods can be employed to prepare NCs containing
multiple drugs employing any drug-polymer conjugate made by the
methods herein. The methods can also be employed to prepare NCs
where the core and shell are made from different polymer conjugates
with the same drug e.g. Ptxl-LA.sub.200/Ptxl-LA.sub.100, or
Ptxl-LA.sub.100/Ptxl-LA.sub.200. In the above methods the relative
amounts of polymer conjugate can be varied as desired to obtain
particle so desired size and properties.
[0172] FIG. 10B is a graph showing the change in particle size of
NCs on addition of different polymer conjugates to the NC to form
core-shell NCs.
[0173] Particle sizes for FIGS. 10 A and B were analyzed by dynamic
light scattering. More specifically, particle sizes were detected
by a ZetaPALS dynamic light-scattering (DLS) (15 mW laser, incident
beam=676 nm; Brookhaven Instruments, Holtsville, N.Y.).
Example 16
Cytotoxicity Test for NCs
[0174] PC-3 cells were plated in a 96-well plate for 24 h (10,000
cells per well). On the day of experiments, cells were washed with
prewarmed PBS and different concentration of fresh prepared NCs
(prepared in 1.times.PBS) was added. The control cells were
incubated with medium. The cells were incubated for a total of 24
hours in the incubator at the 5% CO.sub.2 atmosphere. After that,
the medium was removed and reconstitute with MTT solution and
OptiMEM medium for a further incubation of 3 hours. The resulting
crystals were dissolved and final cell viability was assessed
calorimetrically by microplate reader at 655 nm. The results of
certain cytoxicity studies are shown in FIGS. 7A-D for NCs formed
with Ptxl, Dtxl, CPT, and Doxo with varying M/I ratios compared to
free drug as shown in the figure.
[0175] Analogous MTT cytotoxicity tests were performed using NC
carrying multiple drugs in human prostate cancer cells, PC-3 cells,
with 72 hours of incubation. Relative toxicities of a series of NC
as measured by IC.sub.50 is reported in Table 3.
TABLE-US-00003 TABLE 3 MTT Cytotoxicity for Multi-Drug Loaded NCs
NC IC.sub.50 Ptxl-LA.sub.25 837.22 .+-. 29.81
Ptxl-LA.sub.25/CA-LA.sub.25 622.14 .+-. 14.61
Ptxl-LA.sub.25/CA-LA.sub.10 168.01 .+-. 10.79 Doxo-LA.sub.25 286.68
.+-. 5.65 Doxo-LA.sub.25/CA-LA.sub.25 240.16 .+-. 18.36
Doxo-LA.sub.25/CA-LA.sub.10 84.89 .+-. 7.14
[0176] As shown in the results of Table 3, the dual drug NC's
combining the taxane (paclitaxel) and cyclopamine or the
anthracycline antibiotic (doxorubicin) and cyclopamine exhibit
synergistic effect against the prostate cancer cells. With being
bound to any particular theory of activity, it is believed that
inhibition of Shh by cyclopamine substantially improved the
efficacy of the taxane and anthracycline antibiotic against cancer
cells.
Example 17
Inhibition of the Hedgehog Pathway Using NCs Containing Cyclopamine
Assessed Using a Luciferase Assay
[0177] Shh-LIGHT2 cells, which stably incorporated Gli-dependent
firefly luciferase and constitutive Renilla luciferase reporters,
were cultured to confluency in 96-well plates and then treated with
(1) various concentrations of cyclopamine carried in
PLA-cyclopamine NCs (e.g., CA-LA.sub.10 NCs or CA-LA.sub.25 NCs) in
DMEM containing 0.5% bovine calf serum with or without (2)
PLA-purmorphamine NEs. FIG. 11A is a graph of relative Renilla
Luciferase activity as a function of cycloproamine concentration
(microM). The treated cells were then cultured for 36 h under
standard conditions, and firefly and Renilla luciferase activities
were determined using a dual luciferase kit (Promega) according to
the manufacturer's protocols.
Example 18
Effect of Catalyst on Structure of Conjugates
[0178] As described above in Example 1, in cases in which the drug
or other chemical species carried more than one functional group
that can function for initiation of polymerization with a catalyst,
the structure or nature of the catalyst can affect which of the
functional groups is involved in the polymerization and to the site
or sites in the drug (or other chemical species) the growing
polymer attaches. FIG. 12 shows the effect of use of different
catalysts on the molecular weight and polydispersity (PDI) of
drug-polymer conjugates made by the methods herein. In a specific
case, the figure shows the results of using different catalysts on
conjugate formation with pacitaxel using LA/Ptxl/catalyst molar
ratio of 200/1/1 to prepare Pxtl-LA.sub.200 of expected MW of
29,653. Ptxl formally contains three OH groups (2'-OH, 1-OH and
7-OH) that could initiate polymerization of LA. As noted above,
however, it is considered less likely for steric reasons that the
1-OH would be involved in initiation which is believed to involve
metal oxide formation with the OH group. It is believed that a
source of polydispersity in the formation of Pxtl-LA conjugates is
the formation of some portion of the conjugates with polymer
attached at two sites in the molecule. As shown in the figure, the
actual MW and the PDI as measured by standard Gel Permeation
Chromatography of Pxtl-LA.sub.200 conjugates varies as a function
of the catalyst used. Use of Zn catalyst with bulky ligands
(catalysts 4 and 5, from FIG. 12) results in conjugates with lower
polydispersity. It is believed that the lower polydispersity is
associated with increased selectivity for polymer attachment to one
site, likely the 2'-OH site in Ptxl.
Example 19
Determination of MW and PDI of Drug-Polymer Conjugates Using Gel
Permeation Chromatography
[0179] Drug-polymer conjugates were prepared as described in
previous examples employing the drug as the initiator (in the
presence of catalyst) for polymerization of LA. Actual MW and PDI
were measured using well-known methods of Gel Permeation
Chromatography.
TABLE-US-00004 TABLE 4 GPC data of Nanoconjugates containing Ptxl,
CPT, Dtxl, Doxo, CA or CP NC Expected MW Actual MW PDI
Ptxl-LA.sub.200 29653 28160 1.021 Ptxl-LA.sub.150 22453 22490 1.032
Ptxl-LA.sub.100 15253 14030 1.043 Ptxl-LA.sub.50 8053 9658 1.040
Cpt-LA.sub.200 29148 30440 1.176 Cpt-LA.sub.100 14748 20080 1.207
Cpt-LA.sub.75 11148 12670 1.173 Cpt-LA.sub.50 7548 8978 1.257
Cy5-LA.sub.300 43706 44550 1.098 Cy5-LA.sub.200 29306 30790 1.213
CA-LA.sub.150 22011 22620 1.166 CA-LA.sub.100 14811 19800 1.102
Dtxl-LA.sub.300 44006 51350 1.045 Dtxl-LA.sub.200 29606 30600 1.095
Dtxl-LA.sub.100 15206 20700 1.086 Dtxl-LA.sub.50 8006 12090 1.169
CP-LA.sub.200 30002 40150 1.161 CP-LA.sub.100 15602 31810 1.324
CP-LA.sub.50 8402 21440 1.337 Doxo-LA.sub.200 29343 40630 1.156
Doxo-LA.sub.100 14943 19400 1.253
[0180] The forgoing examples are illustrative and in no way are
intended to limit the scope of the invention. [0181] 1. Duncan, R.
The dawning era of polymer therapeutics. Nature Reviews Drug
Discovery 2, 347-360 (2003). [0182] 2. Duncan, R. Polymer
conjugates as anticancer nanomedicines. Nature Reviews Cancer 6,
688-701 (2006). [0183] 3. Haag, R. & Kratz, F. Polymer
therapeutics: Concepts and applications. Angewandte
Chemie-lnternational Edition 45, 1198-1215 (2006). [0184] 4. Kim,
C. J. Effects of drug solubility, drug loading, and polymer
molecular weight on drug release from polyox (R) tablets. Drug
Development and Industrial Pharmacy 24, 645-651 (1998). [0185] 5.
Kataoka, K., Harada, A. & Nagasaki, Y. Block copolymer micelles
for drug delivery: design, characterization and biological
significance. Advanced Drug Delivery Reviews 47, 113-131 (2001).
[0186] 6. Wagner, V., Dullaart, A., Bock, A. K. & Zweck, A. The
emerging nanomedicine landscape. Nature Biotechnology 24, 1211-1217
(2006). [0187] 7. Gradishar, W. J. Albumin-bound paclitaxel: a
next-generation taxane. Expert Opinion on Pharmacotherapy 7,
1041-1053 (2006). [0188] 8. Desai, N. & Soon-Shiong, P. (U.S.
Pat. No. 6,506,405, 2003). [0189] 9. Desai, N. & Soon-Shiong,
P. (U.S. Pat. No. 6,753,006, 2004). [0190] 10. Musumeci, T. et al.
PLA/PLGA nanoparticles for sustained release of docetaxel.
International Journal of Pharmaceutics 325, 172-179 (2006). [0191]
11. Dong, Y. C. & Feng, S. S. Methoxy poly(ethylene
glycol)-poly(lactide) (MPEG-PLA) nanoparticles for controlled
delivery of anticancer drugs. Biomaterials 25, 2843-2849 (2004).
[0192] 12. Avgoustakis, K. et al. PLGA-mPEG nanoparticles of
cisplatin: in vitro nanoparticle degradation, in vitro drug release
and in Vivo drug residence in blood properties. Journal of
Controlled Release 79, 123-135 (2002). [0193] 13. Moghimi, S. M.,
Hunter, A. C. & Murray, J. C. Nanomedicine: current status and
future prospects. Faseb Journal 19, 311-330 (2005). [0194] 14.
Feng, S. S., Mu, L., Win, K. Y. & Huang, G. F. Nanoparticles of
biodegradable polymers for clinical administration of paclitaxel.
Current Medicinal Chemistry 11, 413-424 (2004). [0195] 15. Cheng,
J. et al. Formulation of functionalized PLGA-PEG nanoparticles for
in vivo targeted drug delivery. Biomaterials 28, 869-76 (2007).
[0196] 16. Dechy-Cabaret, O., Martin-Vaca, B. & Bourissou, D.
Controlled ring-opening polymerization of lactide and glycolide.
Chemical Reviews 104, 6147-6176 (2004). [0197] 17. Cheng, M.,
Attygalle, A. B., Lobkovsky, E. B. & Coates, G. W. Single-site
catalysts for ring-opening polymerization: Synthesis of
heterotactic poly(lactic acid) from rac-lactide. Journal of the
American Chemical Society 121, 11583-11584 (1999). [0198] 18.
Pratt, R. C., Lohmeijer, B. G. G., Long, D. A., Waymouth, R. M.
& Hedrick, J. L. Triazabicyclodecene: A simple bifunctional
organocatalyst for acyl transfer and ring-opening polymerization of
cyclic esters. Journal of the American Chemical Society 128,
4556-4557 (2006). [0199] 19. Chamberlain, B. M. et al.
Polymerization of lactide with zinc and magnesium beta-diiminate
complexes: Stereocontrol and mechanism. Journal of the American
Chemical Society 123, 3229-3238 (2001). [0200] 20. Nishiyama, N.
& Kataoka, K. in Polymer Therapeutics Ii: Polymers as Drugs,
Conjugates and Gene Delivery Systems 67-101 (SPRINGER-VERLAG
BERLIN, BERLIN, 2006). [0201] 21. Lavasanifar, A., Samuel, J. &
Kwon, G. S. Poly(ethylene oxide)-block-poly(L-amino acid) micelles
for drug delivery. Advanced Drug Delivery Reviews 54, 169-190
(2002). [0202] 22. Kwon, G. S. & Kataoka, K. Block-Copolymer
Micelles as Long-Circulating Drug Vehicles. Advanced Drug Delivery
Reviews 16, 295-309 (1995). [0203] 23. Farokhzad, O. C. et al.
Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy
in vivo. Proceedings of the National Academy of Sciences of the
United States of America 103, 6315-20 (2006). [0204] 24. Cheng, J.,
Teply, B., Sherifi, I., Langer, R. & Farokhzad, O. Formulation
of Functionalized PLGA-PEG Nanoparticles for In Vivo Targeted Drug
Delivery. Biomaterials 28, 869-876 (2007).
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