Note: Descriptions are shown in the official language in which they were submitted.
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Title: DRUG DELIVERY SYSTEM
~j LE D OF'"HE INVENTION
The present invention relates to a drug delivery system, and more particularly
to
a system which enhances delivery of two or more drugs which act together.
BACKGROUND OF THE INVENTION
Chemotherapy with anti-cancer drugs is usually limited by its chronic
cardiotoxicity, immunosuppressive activity, and necrotic reaction at the
injection site.
Although regional therapy (e.g. intraarterial infusion of anticancer drug into
the arterial
leading to a tumor) has been used to reduce the systemic toxicities, high
local toxicity is
still a problem. There is a need to improve current chemotherapy to reduce
toxicity of the
chemotherapeutic agents.
Attempts have been made to reduce the toxicity of chemotherapeutic agents by
incorporating the agents into a delivery vehicle to target to a particular
site. Typically,
drug delivery vehicles are formed as aqueous carriers, gels, polymeric
material inserts, or
particulates incorporating the agent. Once the drug delivery vehicle is placed
at a
desired site, the agent is released over a prolonged length of time. The time
release of the
agent will depend on factors such as the release mechanism of the agent from
the drug
delivery vehicle (e.g. erosion or diffusion), the amount of agent in the drug
delivery
vehicle, the solubility of the drug in the surrounding physiological medium,
and in the
case of particulate delivery vehicles, the particle size or size distribution
of the vehicle.
A drug delivery system is desired that enhances delivery of the
chemotherapeutic agent to the target organ and prevents loss of the agent in
the efferent
venous drainage from the organ. Microspheres and microcapsules, (collectively
referred to
as "microparticles") have been described for delivery of active agents to
target organs.
Due to their size, they are trapped in the microvasculature of tissues when
administered
via the regional arterial, where they release their payload. This process is
referred to as
"chemoembolization". Chemoembolization has been reported to be useful in the
treatment of inoperable liver tumors, pain control of bone lesions, as a
preoperative
adjuvant for locally invasive tumors, and in the treatment of solid tumors in
liver, kidney,
breast, lung, and head and neck (T. Kato, Microspheres and Regional Cancer
Therapy,
CRC Press, Boca Raton (1994)). Animal and human pharmacokinetics studies have
shown
enhanced drug exposure to tumors and diminished systemic toxicity by
chemoembolization
as compared with regional organ perfusion of the free drugs.
In one study, microspheres prepared using ion-exchange principles were
reported
to exhibit high doxorubicin loading capacity (>30%), whereas those using
chemical cross-
linkage and physical entrapment approaches, display drug levels less than 15%
(N.
Wilmott and J. Daly, Microspheres and Regional Cancer Therapy, CRC Press, Boca
Raton
SUBSTITUTE SHEET (RULE 26)
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(1994)). Besides high loading capacity, ion-exchange microspheres were also
reported to
provide sustained drug release profiles so that the exposure of the tumor to
the drug can be
maximized (Codde et al Anticancer Research. 13(2):539-43 (1993)). In
comparison with
controls, DOX treatment with free drug, liposomes or microspheres
significantly reduced ,
tumor growth by 56% (P < 0.001), 51% (P < 0.01) and 79% (P < 0.001)
respectively.
Furthermore, the DOX-microsphere treatment was reported to be significantly
better .
than either of the other DOX treatments (53%, P < 0.05) or the sham-
microsphere treated
group (64%, P < 0.05).
The effectiveness of chemotherapy has also been limited by the development of
a multidrug resistance (MDR) phenotype in cancer patients treated chronically
with
certain chemotherapeutic agents. One of the major mechanisms of multidrug
resistance
has been the over-expression of an energy-dependent transport system i.e. P-
glycoprotein
{P-gp) on tumor cell membranes. Chemosensitizers which reverse MDR usually by
interaction with P-gp have been concurrently administered with
chemotherapeutic agents
to improve chemotherapy. Promising results have been reported in some non-
responsive
tumors following concurrent administration of a chemotherapeutic agent and a
chemosensitizer. However, systemic toxicity is frequently increased with the
treatment.
The various drug delivery systems devised for the purpose of chemoembolization
have not considered modulation of MDR, nor have they considered reducing the
side
effects observed with MDR. Therefore, novel formulations are required which
combine
the advantages of targeted delivery and chemosensitization.
BUMMARY OF THE INVENTTON
This invention involves the development of a drug delivery composition
containing biodegradable nano- or microspheres which are chemically modified
with
functional groups to introduce ion-exchange properties to the microspheres,
and to
modulate hydrophobicity, mechanical strength and drug release profiles. The
new
system, similar to a polylactic acid/polyglycolic acid system in terms of
mechanical
strength and drug release profiles, has much higher loading capacity for ionic
chemotherapeutic agents like doxorubicin, vinblastine, verapamil, quinidine
etc..
Therefore, once used in targeted cancer chemotherapy, it is more efficient as
a drug
carrier. Additionally, since loading of single or multiple ionic agents can be
carried out
with ease, for the refractory multi-drug resistant (MDR) cancer phenotypes,
the use of
this system can achieve simultaneous intratumor delivery of both a
chemotherapeutic
agent and a chemosensitizer, resulting in improved therapeutic effects and
much reduced
systemic toxicity. Size control of the new system is easily obtained,
facilitating
confirmation of clinical utility, and large-scale production.
Broadly stated, the present invention relates to a drug delivery composition
comprising microspheres containing at least one chemotherapeutic agent and at
least one
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chemosensitizer wherein the microspheres have a biodegradable polymer matrix
with
functional groups which associate with the chemotherapeutic agent and
chemosensitizer.
The invention also contemplates a method for preparing microspheres containing
at least one chemotherapeutic agent and at least one chemosensitizer
comprising:
(a) obtaining microspheres having a biodegradable polymer matrix with
functional groups which associates with a chemotherapeutic agent and
chemosensitizer; and
(b) mixing the microspheres with at least one chemotherapeutic agent and at
least one chemosensitizer.
The invention also provides a method for treating multidrug resistant tumors
in a
subject comprising administering to the subject an effective amount of a drug
delivery
composition comprising microspheres containing at least one chemotherapeutic
agent and
at least one chemosensitizer wherein the microspheres have a biodegradable
polymer
matrix with functional groups which associate with the chemotherapeutic agent
and
chemosensitizer.
Other features and advantages of the present invention will become apparent
from the following detailed description. It should be understood, however,
that the
detailed description and the specific examples while indicating preferred
embodiments
of the invention are given by way of illustration only, since various changes
and
modifications within the spirit and scope of the invention will become
apparent to those
skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure la is a graph showing the dynamics of verapamil sorption into
microspheres through ion exchange with an initial concentration of verapamil
at 0.025
mg/ml.
Figure 1b is a graph showing the effect of the microsphere/drug ratio on the
equilibrium level of drug loaded and the yield of drug loading with an initial
verapamil
concentration of 0.05 mg/ml.
Figure lc is a graph showing competitive loading of vinblastine and verapamil.
Figure 2a is a graph showing fractional release of verapamil and vinblastine
from single-agent-loaded microspheres as a function of time.
Figure 2b is a graph showing fractional release of verapamil and vinblastine
from dual-agent-loaded microspheres as a function of time.
Figure 3 is a photograph of freeze-dried CMDEX microspheres loaded with
doxorubicin. Shown by a confocal fluourescent microscope.
Figure 4 is a graph showing release of doxorubicin from microspheres into a
phosphate buffer solution (pH 7.4) at 37 degrees C, as a function of time.
Figure 5 is a graph showing release of verapamil from microspheres in Pluronic
F-127 gel into a phosphate buffer solution (pH 7.4) at 37 degrees C, as a
function of time.
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Figure 6 is a graph showing release of verapamil from hydrophobically
modified microspheres in corn oil and from unmodified microspheres in corn oil
into a
phosphate buffer solution (pH 7.4) at 37 degrees C, as a function of time.
Figure 7 is a graph showing the release of quinidine from CMDEX microspheres
and CMDEX microspheres coated with hydrophobic polymers as a function of time.
Figure 8 is a graph showing vinblastine uptake by parent and MDR CHO cells as
a function of time.
Figure 9 is a graph which depicts the uptake of doxorubicin by multidrug-
resistant murine tumor cells in the presence of chemosensitizing agent,
verapamil.
Figure 10 is an IR spectrum of malefic ester of inulin.
Figure 11 is an IR spectrum of inulin.
Figure 12 is an IR spectrum of a copolymer of malefic acid ester of inulin and
methacrvlic acid.
DETAILED DESCRIPTION OF THE INVENTION
Drug Delivery Composition
As hereinbefore mentioned, the present invention relates to a drug delivery
composition comprising microspheres containing at least one chemotherapeutic
agent and
at least one chemosensitizer wherein the microspheres have a biodegradable
polymer
matrix with functional groups which associate with the chemotherapeutic agent
and
chemosensitizer.
The delivery composition of the invention has many other characteristics which
make it particularly advantageous. The microspheres used in the delivery
system are
bio-degradable and are stable in physiological environments. The microspheres
using ion-
exchange principles exhibit high loading capacity for various chemotherapeutic
agents.
This allows an effective dose to be used to produce high levels of local drug
concentration
with consequent greater therapeutic efficacy. Administration of microspheres
with high
drug loading also results in less of the matrix material being co-administered
to the body,
and biological reaction to the matrix material is minimized. The microspheres
also
permit diffusion of the chemotherapeutic agent and chemosensitizer from the
core
through the matrix at a predetermined release rate. The microspheres can also
be
sterilized for use before addition of the chemotherapeutic agents and
chemosensitizers
avoiding degradation of sensitive therapeutics.
Table 1 provides a comparison of the properties of some commercial products
and
a drug delivery composition of the invention.
The microspheres in the drug delivery composition of the invention comprises a
biodegradable polymer matrix. Such biodegradable polymer matrixes may be
comprised
of polyesters, such as for examples, poly(hydroybutyric acid),
poly(hydroxyvalerianic
acid-co-hydroxybutyric acid), poly(lactic acid), poly(glycoiic acid),
poly(lactic acid-co-
glycolic acid), poly{s-caprolactones), poly(e-caprolactone-co-DL-lactic acid);
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polyanhydrides, for example, poly{malefic anhydride); polyamides such as for
examples
albumin, pol(hydroxyalkyl)-L-glutamines, poly(y-ehtyl-L-glutaminate-co-
glutamic
acid), poly(L-leucine-co-L-aspartic acid), poly (proline-co-glutamic acid;
poly(orthoesters); poly(alkyl 2-cyanoacrylates); and polysaccharides, such as
for
examples, starches, glycogen, dextrans, chitin, glucans, fructans like inulin,
mannans,
xylans, arabinans, galactans, gaiacturonans, xyloglucans, galatomannans,
glucomannans,gaiactogiucomannans, arabinogalactans, carrageenans, agar,
agarose, pectic
acids, pectinic acids, alginic acids alginate, gum tragacanth,
glycosaminoglycans,
hyaluronic acid, chondroitin sulphates, karatan sulphate, dermatan sulphate,
and
heparin; and including bacterial polysaccharides like lipopolysaccharide,
peptidoglycan, teichoic acids, cellulose and xanthan gum. The biodegradable
polymer
has functional groups which associate with the chemotherapeutic agent and
chemosensitizer. The functional groups have the general formula RI-RZ-polymer
where
Rl is an alkyl group, an alkene or alkyne, aryl, alkoxy, or cvcloalkyl,
preferably a C1 to
Cio alkyl, preferably R1 may be methyl, ethyl, 1-propyl, 2-propyl, 1-butyl,
propene,
butene, cyclohexene, methylcyclopropyl, methylcyclohexyl, cyclobutyl, or O-
methyl; R2
is a carboxy group (-COOH), a suifonyl group (-S03), or -NR3R4R5 wherein R3,
R4, and R5
are the same or different and represent hydrogen, alkyl, aryl, or cycloalkyl
preferably
alkyl. R1 R3, R4, and R5 may contain other chemical functional groups such as
halogen,
hydroxyl, amine, amide, nitro, and thiol. In preferred embodiments of the
invention R1 is
methyl and R2 is a carboxy group i.e. the functional group is carboxymethyl.
Ionic chemotherapeutic agents are suitable for use in the delivery composition
of
the invention i.e. either cationic or anionic agents. Examples of ionic agents
which may be
used in the delivery composition of the invention are alkaloids such as
vinblastine and
vincristine, antibiotics such as mitomycin C, doxorubicin (adriamycin),
daunorubicin, and
their derivatives, hormonal agents such as tamoxifen. Other chemosensitzers or
G-
glycoprotein inhibitors include LY-335979 (Eli Lilly) and GW-918
(GlaxoWeiicome).
Ionic chemosensitizers are suitable for use in the delivery composition of the
invention. Examples of suitable ionic chemosensitizers which may be used in
the delivery
system of the invention include calcium channel blockers e.g. verapamii,
nifedipine,
nicardipine, diltiazem, depridil, felodipine, and their derivatives,
calmodulin
antagonists e.g. trifluoperazine and chlorpromazine, antibiotics and analogs
e.g.
cefoperazone and ceftriaxone, indole alkaloids e.g. quinidine, quinine, and
quinacrine, and
the like.
The biodegradable polymer of the microspheres used in the drug delivery
composition of the invention may also have free hydroxyl groups converted to
esters. This
conversion increases the hydrophobicity of the microspheres. Modification of
the free
hydroxyl groups results in increased mechanical strength and slows the drug
release of the
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microspheres in the drug composition. The microspheres can also be coated with
hydrophobic polymers to further increase hydrophobicity. Examples of such
polymers
include poly{methyl methacrylate-co-methacrylic acid), polyurethane, chitin;
poly{hydroxyvalerianic acid-co-hydroxybutyric acid), poly(lactic acid),
poly(glycolic
acid), poly(lactic acid-co-glycolic acid),poly(e-caprolactones), poly{e-
caprolactone-co-
DL-lactic acid); polyanhydrides, e.g., poly(maleic anhydride); polyamides,
e.g.,
albumin, pol(hydroxyalkyl)-L-glutamines,poly(y-ehtyl-L-glutaminate-co-glutamic
acid),poly(L-leucine-co-L-aspartic acid), poly(proline-co-glutamic acid);
poly(orthoesters) ; and poly(alkyl 2-cyanoacrylates).
The microspheres in the drug delivery composition of the invention have a
diameter of between about 300 microns (~tm) and 50 nanometers, and are
preferably 5 to 300
ltm, most preferably 40 to 200 ~tm.
The microspheres in the drug delivery composition of the invention may also
contain inert excipients commonly used to improve the characteristics of the
composition.
For example, inert excipients such as vegetable oil, arachis oil, coconut oil,
maize oil,
almond oil, sesame oil, peanut oil, cottonseed oil, Caster oil, corn oil,
olive oil; thermal
gels such as Pluoronic series; alcohols like benzyl alcohol, ethanol; syrup;
esters such as
ethyl oleate, isopropyl myristate, glycerol, propylene glycol, liquid
macrogols, esters,
may be added to improve viscosity, tonicity, biocompatibility, and release
profile and
the like. The microspheres may also be dispersed in a physiological medium
such as
saline.
Process for Pr~paring_Drue Delivery Composition
A method for preparing microspheres containing at least one chemotherapeutic
agent and at least one chemosensitizer is provided comprising:
(a) obtaining microspheres having a biodegradable polymer matrix with
functional groups which associates with a chemotherapeutic agent and a
chemosensitizer; and
(b) mixing the microspheres with at least one chemotherapeutic agent and at
least one chemosensitizer.
The microspheres having a biodegradable polymer e.g. albumin or dextran, may
be prepared using conventional methods or they may be obtained from commercial
sources.
For example, commercial cross-linked dextran microspheres (Sephadex G-10, G-
25, G-50,
G-100 and G-200) may be obtained from Pharmacia. Preferably the microspheres
have a
particle size of about 40-200 ~.m and a selected pore size range for molecules
with
molecular weight of 100-600,000 Da. The microspheres are chemically modified
to
introduce the functional groups. By way of example, carboxymethylation or
sulphonation
may be carried out to introduce a carboxymethyl group or a sulfonyl group,
respectively. In
an embodiment, carboxymethylated dextran ion exchange microspheres are
prepared by
suspending dextran gel (about 2 to 5 grams, preferably 3 grams) in about 40
wt% sodium
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hydroxide and adding chloroacetic acid (about 10 to 20 grams, preferably 16
grams).
Anionic carboxylic groups (1.95 meq/g) may also be introduced by a base-
catalyzed
reaction ~~ith succinic anhydride.
The present inventors have found that drug loading of the chemotherapeutic
agent and chemosensitizer is proportional to the extent of functional
modification of the
polymer matrix. Generally, drug loading increases with increased modification
of the
microspheres. in an embodiment of the invention, at least 10% of the hydroxyl
groups on
the polymer matrix are modified to provide for at least 50% loading of the two
drugs.
Free hydroxyl groups on the prepared microspheres may be converted to esters,
or
ethers or otherwise blocked, by, for example, introducing fatty groups to OH
groups via an
ester bond, ether bond,amide bond, imide bond. It is also possible to increase
hydrophobicity by introducing hydrophobic polymers by copolymerization or
grafting/block copolymerization or introducing cross-linking agents. Such
further
modification of the free hydroxyl groups results in increased mechanical
strength and
slows the drug release of the microspheres in the drug composition.
It will be appreciated that the polymer matrix of the microspheres will be
modified to a degree to provide an appropriate loading, and release for the
particular
combination of chemotherapeutic agent and chemosensitizer and to suit a
particular
therapeutic application.
Free flowing drug loaded microspheres may be obtained after incubation of the
prepared microspheres in distilled water or a polar organic solvent, such as
ethanol or
methanol (for hydrophobic drugs such as cyclosporin DMSO may be used),
containing the
chemotherapeutic agent and chemosensitizer overnight, followed by filtration,
washing
with distilled water, and freeze-drying. Chromatographic methods may also be
used to
enhance loading of the chemotherapeutic agent and chemosensitizer.
As discussed above, inert excipients commonly used to improve the
characteristics of the composition such as thickeners, surfactants etc. may be
added to the
microspheres to improve viscosity, tonicity, biocompatibility, stability and
the like. In
addition, lubricants, dyestuffs, sweetners, flavouring agents, inert
excipients,
preservatives etc. may be added to the microspheres to improve and
permeability
properties. The microspheres may also be dispersed in a physiological medium
such as
saline.
. The microspheres may be sterilized e.g. by heat or UV light before addition
of
the chemotherapeutic agents and chemosensitizers. The agents and
chemosensitizers may
be sterilized separately avoiding possible degradation of sensitive
therapeutics.
A time release profile for the drugs can be optimized taking into
consideration
the amount of drug loaded into the drug delivery composition, the solubility
of the drug in
the surrounding physiological milieu the affinity of the drug to the
microspheres, the
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hydrophobicity of the microspheres and the coating, and the particle size and
size
distribution of the microspheres in the composition.
Applications
TMe drug delivery composition of the invention exhibits a sustained drug
release
profile, and it provides a chemosensitization effect in drug resistant cells.
Accordingly,
the drug delivery system may be used in the treatment of multidrug drug
resistant tumor
cells. It is expected that the drug delivery compositions will be particularly
useful in the
treatment of malignancies including multiple myeloma, breast cancers, ovarian
cancers,
childhook neuroblastoma, leukemia, pancreas carcinomas, liver carcinomas,
cervical
carcinoma, endometrial carcinomas, and adenocarcinomas of the kidney and
colon. The
drug delivery composition may also be used to deliver other combination drug
therapies,
for examples, AZT and 3TC (anti-AIDS); angiostatin and endostatin
{anticancer); Intro-
A {Schering-Plough) and Ribavirin (ICN) (Hepatitis C treatment); anticancer
drugs (e.g.
5-FU) + vasoconstrictors (e.g.epinephrine); anticancer drugs (e.g. taxol)-~
chemosensitizers
(e.g. cyclosporins). In vitro and in vivo model systems may be used to assess
the
therapeutic efficacy and system. For example, an in vitro system using
multidrug
resistant CHRCS CHO cells may be used to test therapeutic efficacy in
multidrug resistant
tumors.
Therefore, the present invention contemplates a method for treating multidrug
resistant tumors in a subject comprising administering to the subject an
effective amount of
a drug delivery composition comprising microspheres containing at least one
chemotherapeutic agent and at least one chemosensitizer wherein the
microspheres have
a biodegradable polymer matrix with functional groups which associate with the
chemotherapeutic agent and chemosensitizer.
The drug delivery compositions may be delivered to a target site through a
variety of known routes of administration. For example, a drug delivery
composition
comprising cross-linked dextran microspheres for use in treating a multidrug
resistant
tumor may be administered by intratumor injection. Other administration routes
include
oral deliver for verapamil and other weak acidic ox basic drugs; rectal
delivery and
topical adminstration such as creams for dermal or transdermal application,
ophthalmic
application, containing drugs such as actibiotics, antifungal agents,
anticancer drugs,
antiglucoma agents, localanesthetic agents, anti-inflammatory, analgesic
agents.
The drug delivery compositions of the invention can be intended for
administration to humans or animals. Dosages of the chemotherapeutic agent and
chemosensitizer incorporated in the drug delivery composition will depend on
individual
needs, on the desired effect and on the chosen route of administration.
In yet another application, the microspheres loaded with chemosensitizers and
radioiabeled P-glycoprotein substrates (e.g. vinblastine, vincrestine, taxol
and sestamibi
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are injected intratumorally, among which sestamibi is preferable because it is
not a
cytotoxic agent. Radio image of the tumor gives the concentration of the
radiolabeled
chemicals in the tumor. The MDR tumors will show low concentration of the
chemicals iri
the absence of chemosensitizers, whereas the non-resistant tumors higher
concentration.
More importantly, in the presence of microsphere-delivered chemosensitizers,
the
increment of concentration of the radiochemicals will be much higher in the
MDR tumors
than that in the non-resistant tumors.
The following non-limiting examples are illustrative of the present invention:
Examples
Exam 1
Synthesis and Characterization of Dextran Ion Exchange Microspheres
a) Carbox~methylation of Dextran Microspheres (DMDEX/MSl
Cross-linked dextran gels (Sephadex G-200) are used to prepare
carboxvmethylated dextran ion exchange MS drug carriers. In a typical
preparation, 3 g
of dextran gel were suspended in 50 ml of 40 wt.% sodium hydroxide solution
and 16 g of
chloracetic acid were then added to this suspension. The reaction mixture was
gently
agitated for 12 hours at room temperature. The reaction mixture was washed
extensively
with distilled water and then freeze dried. The content of ion exchange
carboxymethyl
groups was assayed by acid-base titration method.
b) Drug Loading and Release Studies
Drug loading is carried out by mixing drug solutions in distilled water with
freeze-dried resins. In a typical preparation, 0.1 g of ion exchange resin was
added to 10
ml of 1°n verapamil or doxorubicin aqueous solution. After overnight
incubation, the resin
was isolated by either centrifugation or filtration followed by extensive
washing with
distilled water and then lyophilization. Unbound drug in the wash was
determined by
UV/VIS spectroscopy (HP8452A). For drug release experiments, the drug-loaded
resins
were added to buffer solution directly, or incorporated into other
pharmaceutical vehicles
first, and then added to the buffer solution. The pharmaceutical vehicles
include aqueous
system such as thermal gels and hydrocarbon system such as sesame oil,
vegetable oil, and
corn oil. UV/VIS spectroscopy was applied to assay the drug released from the
delivery
system into the buffer solution. When more than one compound (i.e.,
doxorubicin or
vinblastine and verapamil} is used for simultaneous loading and release, the
same
loading procedure is followed but the different compounds are analyzed by high
performance liquid chromatography (HPLC).
In a typical loading process, 0.05g of the dry, ionic microspheres (e.g. MG-
50)
were added to 10 ml of 0.5% verapamil aqueous solution: After incubation at
room
temperature for predetermined time intervals,
the microspheres were separated by centrifugation, and the drug concentration
in the
supernatant was analyzed by UV/VIS spectrophotometer (HP 8452A) at a
wavelength of
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270 nm for vinblastine and 278 nm for verapamil. Drug loading was calculated
from the
difference between the amount of drug originally used and that after
incubation with the
ion exchange microspheres. In the case of loading of dual agents, e.g.,
vinblastine and
verapamil, the same procedures were applied except for that the drug assay was
carried
out by HPLC (Waters). The mobile phase consisted of phosphate buffer (ionic
strength 0.1
M, pH7.0), tetrahydrofuran, and methanol with a volume ratio of
0.43:0.41:0.16.
Vinblastine and verapamil were separated in a reverse-phase column (Norva-pak
C-18,
Waters) by the mobile phase at a flow rate of 0.6 ml/min. Their retention time
was 5.94
and 6.82 min., respectively, as measured by a L1V detector at a wavelength of
270 nm.
Curve A in Figure la depicts the fraction of remaining verapamil in the
solution
as a function of time for the microspheres incubated in 0.025 mg/ml verapamil
solution. A
rapid decrease in the remaining drug is observed in the initial 10 hours
followed by a
slower change in the subsequent 10 hours. A plateau is reached after 20 hours
indicating an
equilibrium state. Similar trend is also observed with vinblastine. Therefore,
30 hours of
incubation was carried out for all drug loading to ensure completion of the
process. Curve B
in Figure la shows that the fraction of drug loaded into the microspheres
follows a
typical first-order sorption kinetics, suggesting that the drug loading is
essentially a
diffusion-controlled process like drug release.
The amount of microspheres relative to drug is an important factor influencing
the equilibrium drug content and the yield of drug loading. However, there has
been little
systematic work in this area. The yield of drug loading and the equilibrium
level of
verapamil loaded are plotted in Figure lb against the ratio of the
microspheres to the
drug (M/D ratio). As the M/D ratio increases, the yield of drug loading
increases while
they equilibrium level of drug loaded decreases. This indicates that, in order
to raise the
equilibrium drug content, one has to sacrifice the loading efficiency.
Therefore, the
compromise approach is to control the conditions in the left region, e.g., M/D
ratio
between 1 and 3. In this case, the drug content reaches --30% with the yield
of drug loading
40-60%.
Competitive loading of dual agents is an indication of relative affinity of
the
drugs to the microspheres which is related to the relative release rate.
Figure 1c shows
that the relative amount of vinblastine in the microspheres is higher than
verapamil.
This explains partly why vinlastine is released more slowly than verapamil.
c) Drug release from microspheres in a buffer solution
At predetermined time intervals, the suspension was centrifuged and the
supernatant was analyzed spectroscopically or chromatographically. Figure 2a
shows the
fractional release of verapamil and vinblastine from individually-loaded
microspheres.
Figure 2b depicts the release profiles of verapamil and vinblastine from
dual-agent-loaded microspheres. Both graphs indicate that the drugs are
released for a
prolonged period of time and the release rate of verapamil is higher than that
of
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vinblastine. Figure 3 is a confocal fluorescent photograph of doxorubicin-
loaded
microspheres. Figure 4 illustrates the sustained release of doxorubicin from
the
microspheres into pH 7.4 buffer solution at 37 °C.
d) Drug release from microspheres in a synthetic thermal gel (Pluronic F-127)
An aqueous solution (20-30%) of Pluronic F-127 was prepared at 4
°C. This
solution experiences gelation at temperatures higher than 10 °C.
Microspheres (Sephadex
SP C-25) loaded with ionic drugs) (e.g., verapamil or doxorubicin) were added
to the cold
solution under stirring. The suspension showed the same reversible thermal
gelation as
the parent Pluronic F-127 solution, as demonstrated in heating and cooling
cycles. It was
syringe-injectable at lower temperature. Therefore, once localized at sites of
physiological conditions, it became semi-solid gel. The gel, well tolerated by
tissue itself,
could prevent the microspheres from coagulation and slow down the drug release
rate. In
vitro release from the above mixture was demonstrated by transferring 1 g of
the gel with
5 wt.% of microspheres loaded with 29 wt.% verapamil into a dialysis tubing
(molecular
weight cut-off: 6000). Drug release from the clamped tubing in 200 ml
phosphate buffer
(0..05 M, pH 7.4) was monitored with UV absorbance at 278 nm. Figure 5
displays the
fractional release of verapamil from the ion exchange resin in Pluoronic F-127
into pH 7.4
buffer at 37 °C which is significantly slower than that directly
contact with the buffer
solution (see Figure 2a).
a ) Drug release from microspheres in vegetable oil
Microspheres loaded with ionic drug (i.e., verapamil or doxorubicin) were
added
to corn oil to provide a syringe-injectable oil formulation. The oil phase
behaved as a
barrier for the ion exchange process required for drug release. Therefore,
slower release
was obtained. Hydrophilic polysaccharide microspheres (e.g., SP C-25) and
their
corresponding hydrophobically-modified product (SP C-25/palmitoyl cholride)
were
tested for their release profiles. The hydrophobically-modified product mixed
well in
the oil while the unmodified one coagulated. Release experiments were
undertaken using
the same method described above. The release profiles are presented in Figure
6.
f ) Modification of the R siQ n Hydro hR obic~ for Better Control in Drug
Release
Kinetics
Hydrophobic resins, therefore less swellable in water and probably with higher
affinity for hydrophobic drugs (such as digoxin, taxol, cyclosporins,
nifedipine, cisplatin,
pentaerythritoltetranitrate, indomethacin, theophylline, AZT, and cipro) by
esterification of the ion exchange resins and/or by coating with hydrophobic
polymers
without jeopardizing the ion exchange capacity. Typically, 3 g of CMDEX/MS was
added
to 100 ml of DMSO containing 15 g of acetyl chloride as esteiification agent
and pyridine
as catalyst. After overnight reaction, the mixture was filtered and the resin
was washed
first with organic solvent and then with distilled water. In another example,
15 g of
palmitoyl chloride in the place of acetyl chloride was used to react with 3 g
of ion
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exchange resin {SP-C25) in 100 ml of DMSO with triethylamine as the catalyst.
The same
reaction condition and purification procedures were employed. More hydrophobic
resins
were prepared by coating the esterified microspheres with hydrophobic polymers
such as
poly(lactic-co-glycolic acid). Free-flowing microspheres of lower degree of
swelling in
water were finally obtained by freeze-drying.
Figure 6 illustrates the results of a comparison of the release of verapamil
from
hydrophobically-modified resin in oil vehicle with that without modification.
Much
slower release of the drug was realized from the hydrophobically-modified
resin. Figure
7 shows the release of quinidine from CMDEX/MS alone (without coating) and
CMDEX/MS coated with hydrophobic polymers (with coating). Again, the results
demonstrate that surface coating of the microspheres with hydrophobic polymers
significantly reduced the drug release rate.
Example 2
In vitro studies of drug accumulation of vinblastine by Chinese hamster ovary
(CHO) cells
a) Tissue cell culture and accumulation of 3H-vinblastine by,~arent and
multidrug
resistant cells in presence of chemos nsitizer
Parent {AuxBl) and multidrug resistant (CHRCS) Chinese hamster ovary (CHO)
cells, originally selected from the parent line for resistance to colchicine
(180 times
relatively resistant to colchicine and 30 times to vinblastine) are grown in
25 cm2 and 75
cm2 plastic tissue culture flasks in alpha minimal essential medium (a-MEM),
containing
10% fetal bovine serum and 0.5% penicillin-streptomycin at 37 °C in an
atmosphere of air
and 5% C02.
For the accumulation studies, a similar methodology as described before
(Bondanan, J. et al. Am. Soc. Nephrol. 5:75-84 (1994)) was used. Cells were
grown on 24
well plates for 3-4 days until they reached confluence. Drug accumulation was
initiated
by the addition of 0.5 ml of Earle Balanced Salt Solution (EBSS) containing 21
nM (1/3
3H-labelled and ?/3 cold) vinblastine sulfate and 14C-mannitol (an
extracellular
marker). Cells were then incubated for 0 to 2 h at 37 °C in an
atmosphere of air + 5% C02.
Drug accumulation by the cells was rapidly stopped by aspirating the media and
by
washing the cells twice with 2 ml of ice-cold 0.16N NaCI. The cells were then
lysed with
1 N NaOH, followed by neutralisation with 2 N HCI, and then counted in a
liquid
scintillation counter. Protein determination was performed by a standard
colorimetric
method well know to those skilled in the art. The accumulation of vinblastine
by the
cells was corrected for the extracellular binding as determined by the
accumulation of 14C
mannitol.
Figure 8 shows the uptake of vinblastine over time for both parent AuxBl
(series
2) and mufti-drug resistant CHRC5 CHO cells (series 1). As can be seen from
Figure 8 the
uptake of vinblastine is greater in the parent cells as compared to the MDR
cells. This
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demonstrates that the CHRC5 CHO cells used in this experiment are in fact
multi-drug
resistant cells.
Initially the accumulation of vinblastine over time by the parent and
resistant
CHO cells is determined in the presence and absence of chemosensitizers (i.e.,
20mM and
50mM cyclosporin A, 50mM quinidine, 50mM verapamil) to verify that the cells
are
retaining MDR properties. Then CHRC5 cells are used to evaluate the
effectiveness of
vinblastine and/or verapamil loaded into the microspheres. The amounts of
loading are
controlled to make sure that final concentrations of vinblastine and/or
verapamil are
similar to conditions using free agents.
Table 2 shows the uptake of vinblastine by the mufti drug resistant CHO cells
in
the presence of immobilized verapamil as compared with free agents and placebo
microspheres. At one hour the uptake of vinblastine is 0.69~0.07 for CMDEX/MS
beads
loaded with vinblastine as compared to 6.81~0.43 for CMDEX/MS beads loaded
with
vinblastine and verapamil. At two hours the uptake of vinblastine is 1.05~0.09
for
CMDEX/MS beads loaded with vinblastine as compared to 8.05~0.64 for CMDEX/MS
beads loaded with vinblastine and verapamil. These results demonstrate that
the
loading of vinblastine is enhanced approximately 9.9 times at one hour and 7.7
times at 2
hours when the microspheres have been loaded with verapamil.
b) Cell viability-Tr~nan blue test
Viability of multidrug resistant (CHRCS) Chinese hamster ovary cells in the
presence chemosensitizers or ion exchange resins is determined according to
the standard
Trypan blue test.
The cytotoxicity of CMDEX/MS or its acetylated derivative AC-CMDEX/MS
was investigated. The results shown in Table 3 demonstrate that CMDEX/MS and
AC-
CMDEX/MS were not noticeably toxic as compared to a control.
Example 3
In vitro studies of drug accumulation of doxorubicin and vinblastine by marine
tumor cells
Marine breast sarcoma cell line EMT6/P (parent) and the resistant variant
EMT6/AR1.0 are used as the model system. The latter was selected by exposure
to
doxarubicin and overexpression of P-gp. Cells are grown in alpha minimum-
essential
medium (a-MEM) with 10% fetal bovine serum and 0.1 mg/mL kanamycin.
When EMT6/P and EMT6/AR1.0 cells are grown in multiwell plates for 3-4 days,
. reaching sub-confluence, drug accumulation is initiated by the addition of
0.5 mL a
MEM/30 mM HEPES containing 14C-doxorubicin and 3H-mannitol (an extracellular
marker). After incubation for 0-120 minutes, cellular accumulation is rapidly
stopped by
aspirating the medium and by washing the cells twice with 1 mL ice-cold 0.9%
NaCl.
The cells are lysed with 0.5 N NaOH followed by neutralization and then
courted in a
liquid scintillation counter. The accumulation of 14C-doxorubicin by the cells
is corrected
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for extracellular binding as determined by the accumulation. The effect of
immobilized
chemosensitizer and/or cytotoxic agent on drug accumulation is investigated
and compared
'with placebo and free chemosensitizers and free cytotoxic agent.
In the studies of vinbiastine accumulation by the murine cells, the same
procedures are employed as in the studies of doxorubicin except that 3H-
vinblastine and
14C_mannitol are used.
Figure 9 depicts the uptake of doxorubicin by the multidrug-resistant murine
tumor cells in the presence of chemosensitizing agent, verapamil, is increased
more than
2.6 fold. Based on these results and the in vitro release rate of doxorubicin
(Figure 4) and
verapamil (Figure 2a), the cellular uptake of doxorubicin loaded in the
microspheres is
expected to increase to the same extent in the presence of verapamil. A
slo~n~er uptake
kinetics may be anticipated because of slow release of doxorubicin from the
microspheres.
Example 4
In vivo therapeutic trials using mice and murine tumor cells
Parent and multidrug resistant murine tumor cells, EMT6/P and EMT6/AR1.0, are
grown in monolayer using the same method described in Example 3. The cells are
released
from the plates with trypsin and resuspended for injection into mice. In vivo
growth is
initiated by intramuscular injection of 1x106 cells into the left hind leg of
8-12 week old
syngeic Balb/c mice ~n~eighing 25-30 g. Growth of tumors is monitored daily by
passing the
tumor-bearing leg through graded holes in a strip of Lucite and the leg
diameter is
converted to an estimate of tumor weight using a previously defined
calibration curve.
When mouse tumors have reached a size of 0.6-0.9 g, the therapy is initiated.
Four groups
of 6 mice for each of the two tumor types (EMT6/P and EMT6/AR1.0) are used for
the
therapeutic trials. Each treatment involves intratumoral injection in a volume
of 50-100
mL containing 10-2~ mg chemotherapeutic agent and chemosensitizer. Group C
serves as
control and receives placebo microspheres. Group D receives doxorubicin-loaded
microsphere, Group V receives verapamil-loaded microsphere, and Group DV
receives
both doxorubicin- and verapamil-loaded microspheres. Tumor growth is monitored
daily
and the mice are sacrificed when tumors reach an estimated size of 1.5 g
(about 6% of body
weight). The dosages of doxorubicin and verapamil is determined from in vitro
data and
preliminary in vivo experiments.
Tumor response to therapy is assessed using the growth curves and the delay in
growth of tumors indicates therapeutic response.
Example 5
Synthesis and characterization of microspheres of malefic ester of inulin and
methacrylic
acid
Preparation of malefic ester of Inul'n
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g of inulin was dissolved in 200 ml of warm water (-40 °C). 12 g of
malefic
anhydride and 3 g of sodium acetate were added. The reaction mixture was
stirred for 24
hours at 40 °C. The reaction product was isolated by precipitation in a
4-fold volume of
anhydrous ethyl ether. The precipitates were washed with ice cold water,
dissolved in
5 an aqueous solution of sodium bicarbonate. The sodium salt of the product
was
precipitated by addition of acetone, filtered, and then dissolved in water.
Further
purification was undertaken by precipitating the free ester with hydrochloric
acid,
washed with ice cold water (0-5 °C), and then dissolved in 4:1 (V/V)
mixture of acetone
and isopropyl alcohol. The product was finally separated by addition of ether.
The
resultant malefic half ester of inulin was a white powder, soluble in
methanol, ethanol,
DMF, swollen by cold water, soluble in Harm water, and insoluble in ether.
characterization of malefic ester of Inulin
Infra red spectra
Figure 9 shows the FTIR spectrum of malefic half ester of inulin in comparison
with that of inulin (Figure 10). C-O stretching band, i.e., 1100-1300 cmn in
the ester bond
is clearly seen in the figure, which is absent in Figure 10. The presence of
malefic ester is
indicated by strong C=O stretching absorption band in the region of 1730 cm 1
and that of
olefinic double bond in conjugation with carbonyl group at 1640 cm 1 indicate.
The broad
and intense O-H stretching absorption at 3350 cm-1 in inulin is significantly
reduced after
esterification.
Copolvmerization of inulin half ester of malefic acid H~ith methacrvlic acid
Copolymerization was carried out in distilled water at 70 °C under
nitrogen
atmosphere using N,N-methylene bisacrylamide (BIS) as the cross linking agent
and
potassium persulfate (KPS) as the initiator. To monomer mixture containing
malefic half
ester of inulin and inhibitor-free methacrylic acid to a total concentration
of 1.82 mole,
10% BIS was added and then polymerization was initiated by the addition of
small
amount of concentrated KPS 0.05 mole solution. After polymerization for 4
hours, the
copolymer was separated and washed several times with distilled water to
remove the
impurities.
Infra red spectra
Figure 11 is the IR spectrum of the copolymer of malefic ester of inulin and
methacrylic acid which was synthesized without using cross-linking agent. It
shows
absence of olefinic double bond at 1620 cm-1 (refer to Figure 9) indicating
that the double
bonds of malefic half ester of inulin are involved in copolymerization.
Solubility of the copolymer
The solubility of the copolymer and inulin ester was tested in order to verify
the
incorporation of the monomer into the copolymer. The results are shown in
Table 4
together with the solubility of the monomers. Taking into account that
monomers are
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soluble in water, DMF, and methanol, the solubility change indicates that most
of
monomers have been incorporated into the copolymer.
Degree of swelling
The degree of swelling of the cross-linked copolymer of malefic half ester of
inulin and methacrylic acid was determined by allowing the copolymer sample to
equilibrate in distilled, deionized water at room temperature. The mass of the
swollen
sample was determined by blot-and-weigh technique. Dry copolymer mass was
determined by drying the sample at room temperature and then in vacuum at room
temperature. The degree of swelling was then calculated by the ratio of the
weight of
swollen copolymer to that of dry copolymer.
Preparation of microspheres of malefic ester of inulin and methacrylic acid
The microspheres of malefic ester of inulin and methacrylic acid were prepared
by suspension polymerization. Aqueous solution of inulin ester, methacrylic
acid, and
N,N-methylene bisacrylamide was dispersed in oil phase by vigorous stirring.
In a
typical polymerization, 10 ml of the aqueous solution was added to 100 ml of
paraffin oil
containing 10% (v/v) nonionic surfactants, e.g., Pluoronic L-62 and Span 80
with a volume
ratio of 3:1. The polymerization was initiated at 70 °C under nitrogen
atmosphere by
addition of potassium persulfate as the initiator. After polymerization for 6
hours, the
microspheres were filtered and washed with distilled water to remove the
impurities.
The purified microspheres containing ion exchange groups, -COO-, were subject
to the tests
of drug loading and release, in vitro evaluation of drug uptake, and in vivo
therapeutic
trials using the same methods as for dextran-based microspheres.
Exam a 6
Sustained retention of doxorubicin in marine solid tumors
The mouse tumors were grown using the method described in Example 4. When
the tumors reached a size of 0.6-0.9 g, 50-100 mL of microsphere suspension
was injected
into the tumor. Typically, 50 mL of the suspension containing 10 mg/uL
microspheres
loaded with 60% doxorubicin was administered. The mice were sacrificed at
various time
intervals (e.g., 3 hours, 1 day, and 3 days) and the tumors were taken and
immersed in
liquid nitrogen and then in formaline to fix the texture of the tissue. The
tumors were cut
into thin slices which were then subject to confocal fluorescence imaging. The
fluorescent
image of doxorubicin was obtained using confocal fluorescent microscopy (MRC
600) with
an excitation wavelength of 488 nm and an emission wavelength longer than 515
nm.
Based on personal observation, the tumors containg the microspheres were still
releasing
doxorubicin.
While the present invention has been described with reference to what are
presently considered to be the preferred examples, it is to be understood that
the invention
is not limited to the disclosed examples. To the contrary, the invention is
intended to
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cover various modifications and equivalent arrangements included within the
spirit and
scope of the appended claims.
All publications, patents and patent applications are herein incorporated by
reference in their entirety to the same extent as if each individual
publication, patent or
patent application was specifically and individually indicated to be
incorporated by
reference in its entirety.
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Table 1. Comparison of Commercial Products and Drug Delivery Composition of
the
Invention
Ion- High Drug Mech an- Biodegra Size Hydroph
exchange capac- Releas i c a 1 d-abilityC o o-bicity
n-
Microsphereity a Strength trot
s Time
Poly-styreneY Y Y N Y Y
10C'.ross-linkedY N N Y Y N
Dextran
PGA/PLA* N Y Y Y N Y
Our System Y _ Y Y Y Y VARIES
*Represents microspheres made in situ for controlled release formulation.
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Table 2. Vinblastine uptake by CHRCS cells in the presence of chemosensitizers
Time Sample Vinblastine Uptake Ratio of
( mole/m rotein/ml) Enhancement
1 h Control 1.560.11
Blank Beadsl 1.02_+0.05 0.65
Verapamil(50uM) 5.810.52 3.7
Ver(50uM)+BBl 5.780.69 3.7
VEbead2 5.090.61 3.3
Cys A(20uM) 12.771.36 8.2
Cys A(20uM)+BBl 12.401.32 7.9
MS13 0.690.07 0.44
MS24 6.810.43 4.4
MS2 / MS1 9. 9
2 h Control 2.080.09
Blank Beadsl 1.160.19 0.56
Verapamil(50uM) 6.120.55 2.9
Ver(50mM)+BB1 6.540.24 3.1
VEbead2 5.560.40 2.7
Cys A(20uM) 13.51.62 6.5
Cys A(20uM)+BBl 14.21.98 6.8
MS13 1.050.09 0.51
MS24 8.050.64 3.9
MS2 / MSl 7.7
1. Placebo - blank CMDEX/MS beads (1% wt./v) were added into transport
medium, 50mM verapamil or 20mM Cys A.
l~ 2. CMDEX/MS beads (1% wt./v) loaded with verapamil.
3. CMDEX/MS beads (1% wt./v) loaded with vinblastine.
4. CMDEX/MS beads (1% wt./v) loaded with vinblastine and verapamil.
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Table 3. Cytotoxicity of CMDEX/MS and its acetylated derivative (AC-CMDEX/MS)
Sam le Cell Viability
(Nonviable/Viable,
%)
1 Hour Incubation3 Hour Incubation
_
Control 82 1914
CMDEX/MS 7t1 2116
AC-CMDEX/MS 82 20f4
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Table 4. Solubility behavior of monomers and copolymer
Methanol DMF Water
MAA + + +
Malefic half + + +
ester
of inulin
Copolymer - - ~ -
+ soluble; - insoluble, * swelling.
SUBSTITUTE SHEET (RULE 26)
