Note: Descriptions are shown in the official language in which they were submitted.
CA 02387925 2002-04-18
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ivIAGNETIC TARGETED CARRIER CO1~IPOSED OF IRON AND POROUS
MATERIALS FOR THE TARGETED DELIVERY OF BIOLOGICALLY ACTIVE
AGENTS
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the United States national phase of International Patent
Application No. PCT/LJS00/ , filed October 13, 2000, which claims priority to
United
States Provisional Application Serial No. 60/160, 293, filed October 18, 1999.
INTRODUCTION
This invention relates to compositions, methods of manufacture and methods for
delivery of biocompatible particles to a selected location in a body and, more
particularly,
relates to particles capable of carrying biologically active compounds and
which provide for
targeted magnetic transport of the particles and their maintenance in a
predetermined place as
a localized therapeutic treatment for disease, diagnostic aid, or bifunctional
composition
capable of acting as both a diagnostic and therapeutic went.
The site-specific delivery of biologically active agents would enable
enhancement of
therapeutic activity of chemotherapeutics while minimizing systemic side
effects. Magnetic
carrier compositions for treating various disorders have been previously
suggested and
utilized, and include compositions which are guided or controlled in a body in
response to an
externally applied magnetic field. (See Lieberman et al., U.S. Patent
4,849,209; Schroder et
al., U.S. Patent 4,501,726; Chang, U.S. Patent 4,62,257; and Mirell, U.S.
Patent 4,690,130).
One such known composition, deliverable by way of intravascular injection,
includes
microspheres made of a ferromagnetic component covered with a biocompatible
polymer
(albumin, gelatin, and polysaccharides) which also contains a drug (Driscol,
C.F. et al., Prod.
Am. Assoc. Cancer Res., 1980, p. 261).
It is possible to produce albumin microspheres up to 3.0 ~,~m in size
containing a
magnetic material (magnetite Fe30a) and the anti-tumoral antibiotic
doxontbicin (Widder, K.
et al., J. Pharm. Sci., 68:79-82, 1979): Such microspheres are produced
through thermal
and/or chemical denaturation of albumin in an emulsion (water-in-oil), with
the disperse
1
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/2861~
phase containing a magnetite suspension in a medicinal solution. A similar
technique has
been used to produce magnetically controlled, or guided, microcapsules covered
with
ethylcellulose containing the antibiotic mitomycin-C (Fujimoto, S. et al.,
Cancer, 56: 2404-
2410,1985).
Magnetically controlled liposomes, 200 nm to 800 nm in size. capable of
carrying
preparations that can dissolve atherosclerotic formations are also known. This
method is
based upon the ability of phospholipids to create closed membrane structures
in the presence
of water (Gregoriadis G., Ryman B.E., Biochern. J., 124:58, 1971).
Such previously known compositions have not always proven practical and/or
effective. Often, there is ineffective drug concentration delivered to the
targeted site. Many
of the compositions lack adequate transport capacity, exhibit weak magnetic
susceptibility,
and/or require extremely high flux density magnetic fields for their control.
In some cases,
there is no real localization of the particles enabling a precise local
therapy. Other
shortcomings include non-specific binding and toxicity to untargeted organs
for compositions
incorporating antibodies and peptides, and drug diffusion outside of the
desired site for intra-
tumoral injection based technologies. Some compositions are difficult to
manufacture or
prepare consistently, sterilize, and store without changing their designated
properties.
Thus, there remains a need for an effective biocompatible composition that is
capable
of being transported magnetically and that is relatively easy to manufacture,
store and use.
One suggested composition comprises ferrocarbon particles for use as
magnetically
susceptible material for magnetically controlled compositions. These particles
have a major
dimension (i.e., largest diameter) of about 0.2 um to about 5.0 ~m (and
preferably from
0.5 um to 5.0 um) and contain from about 1.0% to about 95.0% (by mass) of
carbon, with
the carbon strongly connected to iron. The particles are obtained by jointly
deforming (i.e..
milling) a mixture of iron and carbon powders. See U.S. Patents 5,549,915;
5,651,989;
5,705,195 and U.S. Patent Application Serial Nos. 09/003,286, and 09/226,818,
which are
incorporated herein by reference.
Previous applications of this technology arose from a desire to make alloys
that were
not achievable through smelting processes. Not all conceivable alloys can be
made by
smelting, as the solubility of one molten metal in another limits the
concentrations that the
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/2861~
mixtures can achieve. Milled ferrocarbon particles were derived as an
adaptation of a
technique for making alloys. The milling technique is fine tuned to produce a
durable
connection between the two materials without intimately mixing them as an
alloy, which
would result in reduction or elimination of both the magnetic moment and/or
the drug
carrying capacity. The idea of combining iron and carbon by milling arose from
their natural
mixability, as in the smelting process for forming alloys.
SUNIiVIARY OF THE INVENTION
It has now been found that iron-ceramic particles can be produced by the
milling
method. This is surprising because alloys using these materials have not
previously been
demonstrated. Thus, it was not thought that a durable interface between the
iron and the
ceramic material could be formed.
Iron-ceramic composite particles show great versatility to bind to various
drugs that
adsorb at the particle surface for easy incorporation of the active went.
Additionally, iron-
ceramic particles utilize metallic iron with a higher magnetic susceptibility
than iron oxides.
thereby facilitating and expediting mobility to the treatment site.
Furthermore, the
biocompatibility properties of ceramics are well known.
Biocompatible and biodegradable ceramic materials, based on hydroxyapatite and
other calcium phosphate derivative materials have been used as bone
replacement material in
dental and skeletal procedures. However, the concept of magnetically targeting
a ceramic
material used as a carrier is completely novel. This invention provides a
magnetically
responsive composition that carries biologically active substances. Generally,
iron-ceramic
composite particles can be used to target the delivery of a number of
biologically active
agents, diagnostics, or bifunctional compositions. Methods of production and
use thereof are
also provided.
The aim of this invention is to improve some parameters of magnetically
controlled
compositions used for the targeted transport of a biologically active
substance, including:
enabling use of natural bone constituents in the carrier particle, expanding
the categories of
therapeutics and diagnostics for which this technology can be used, increasing
relative
absorption capacity and magnetic susceptibility by, for example, providing a
large number of
CA 02387925 2002-04-18
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ionic Groups that enable binding of compounds by ionic interactions, improving
biocompatibility and biodegradability, intensifying diagnostic and therapeutic
effect,
simplifying the technology of manufacturing the magnetically controlled
composition, and
ensuring its guaranteed long-term storage capabilities without changing the
desired
characteristics.
This is achieved by using suitable composite, iron-ceramic particles, as a
magnetically susceptible material for a magnetically controlled composition.
The particles
are disk and spherically shaped, approximately 0.1 to 10.0 um in diameter, and
contain 1.0%
to 95.0% ceramic (or a derivatized ceramic) and 5.0% to 99.0% iron, by iron.
They are
obtained by jointly deforming (i.e., milling) a mixture of iron and ceramic
powders.
Adsorption occurs on the surface, or modified surface, of the panicle so the
drug is readily
available and capable of incorporation at the treatment site.
The powders are combined in a planetary ball, or attrition mill with a solvent
(e.~.
ethanol). The resulting composite powder is then sieved or magnetically
separated to obtain
the desired fraction of product, and correspondingly, the desired magnetic
susceptibility. The
biologically active agent or diagnostic aid is adsorbed to or deposited on to
the composite
and administered to the patient in a suspension of the composite in a sterile
diluent.
The methods of use include methods for localized in vivo diagnosis or
treatment of
disease providing a magnetically responsive iron-ceramic carrier having
adsorbed thereon a
?p biologically active substance selected for its efficacy in diagnosing or
treating the disease,
and injecting the carrier into the body of a patient. For example, the carrier
is injected by
inserting delivery means into an artery to within a short distance from a body
site to be
treated and at a branch or branches (preferably the most immediate) to a
network of arteries
carrying blood to the site. The carrier is injected through the delivery means
into the blood
?5 vessel. Just prior to injection, a magnetic field is established exterior
to the body and
adjacent to the site with sufficient field strength to guide a substantial
quantity of the injected
carrier to; and retain the substantial quantity of the carrier at, the site.
Preferably, the
magnetic field is of sufficient strength to draw the carrier into the soft
tissue at the site
adjacent to the network of vessels, thus avoiding substantial embolization of
any of the larger
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CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
vessels by the carrier particles. See, for example, U.S. Provisional
Application Ser. No.
60/160,293, which is incorporated herein by reference.
It is therefore an object of this invention to provide a highly magnetically
responsive
composition for optionally carrying biologically active substances and methods
of production
and use thereof.
It is another object of this invention to provide a magnetically responsive
carrier for
biologically active substances that has high magnetic responsiveness, yet is
durable during
storage and use.
It is another object of this invention to provide a magnetically responsive
composition
comprising particles approximately 0.1 to 10.0 um in diameter, each iron-
ceramic panicle
containing 1.0%to 95.0°,% ceramic (or a ceramic derivative) and 5.0% to
99.0% iron, by
mass.
It is still another object of this invention to provide a composition utilized
for
localized in vivo diagnosis or treatment of disease including a carrier with
composite iron-
ceramic particles approximately 0.1 to 10.0 um in diameter, each iron-ceramic
particle
containing 1.0% to 95.0% ceramic (or a ceramic derivative) and 5.0% to 99.0%
iron, by
mass, and having adsorbed thereon one or more optional biologically active
substances
selected for efficacy in diagnosing and/or treating a particular disease.
With these and other objects in view, which will become apparent to one
skilled in
the art from the following description, this invention resides in the novel
construction,
combination, arrangement of parts and methods substantially as hereinafter
described, and
more particularly defined by the appended claims, it being understood that
changes in the
precise embodiment of the herein disclosed invention are meant to be included
as they come
within the scope of the claims.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a magnified photograph (X1000) of composite iron-silica particles.
FIG. 2 is a magnified photograph (X3000) of composite iron-silica particles.
FIG. 3 is a flow diagram of the production process of this invention.
FIG. 4 is a Doxorubicin binding curve for an iron-silica gel composite.
FIG. 5 is Doxorubicin binding curve for an iron-C18 composite.
FIG. 6 is a Scanning Electron Microscopy photograph showing the morphology of
iron-hydroxyapatite particles.
FIG. 7 is the same frame as in FIG. 6, with monitoring of backscatter to show
iron in
white and hydroxyapatite in black.
FIG. 8 is the spectra of the particle shown in FIG. 6 confirming that the
white spot
are composed of iron.
FIG. 9 is the spectra of the particle shown in FIG. 6 confirming that the
black spots
are composed of hydroxyapatite.
FIG. 10 is particle size analysis of hydroxyapatite particles using light
scattering
technique.
FIG.11 is a magnetic susceptibility curve of an iron-hydroxyapatite
microparticle
using magnetometer technique.
FIG. 12 is a Langmuir isotherm plot for iron-hydroxyapatite.
FIG. 13 is a Langmuir isotherm plot for hydroxyapatite (no iron).
FIG. 14 is a doxorubicin desorption profile for iron-hydroxyapatite.
FIG. 15 shows labeling of iron-hydroxyapatite particles with Indium 1 I I by
direct
incubation and stability in different media.
FIG. 16 shows labeling of iron-hydroxyapatite particles with Indium
111/oxyquinoline and stability in different media.
6
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DETAILS OF THE INVENTION
The invention is a composite panicle comprised of 1.0% to 9~.0°,% a
ceramic (or
ceramic derivative) and 5.0% to 99.0% iron, by mass. With compositions having
less than
1.0% ceramic, the binding capacity of a particle is decreased to the point of
being lamely
ineffective for carrying biologically active substances. With compositions of
greater than
9~.0% ceramic, the magnetic susceptibility is generally reduced beyond an
effective level for
targeting biologically active substances in vivo. The particles are disk and
spherically
shaped, approximately 0.1 to 10.0 um in diameter.
The term "ceramic" means a natural or synthetic porous, adsorptive material.
It is
usually, but not necessarily an oxide or mixed oxide, wherein the oxide is
metallic or non-
metallic. It is usually, but not necessarily inorganic. It is usually, but not
necessarily without
a crystalline structure. Examples of synthetic ceramic materials include, but
are not limited
to tricalcium phosphate, hydroxyapatite, aluminum hydroxide; aluminum oxide,
aluminum
calcium phosphate, dicalcium phosphate dihydrate, tetracalcium phosphate,
macroporous
triphasic calcium phosprate, calcium carbonates, hematite, bone meal, apatite
wollastonite
glass ceramics and other ceramic or glass matrices. Also included are polymers
that have a
degree of crystallinity that will support pores and adsorption. Examples of
such polymers
include, but are not limited to polyethylenes, polypropylenes, and
polystyrenes. Appropriate
materials based upon these parameters will be apparanent to any person having
ordinary skill
in the art. A table of examples follows.
Oxide Non-metallic Amorphous
Silica Y Y Y
Hydroxyapatite Y N Y'
Zeolites Y N N
Aluminas Y N Y
Diamond N Y N
Also included in the definition of "ceramic" are silica and silica derivatives
(including, but not limited to octadecycl silane [C~g], octyl silane [C$],
hexyl silane [C~],
phenyl silane [C6], butyl silane [Ca], aminopropylsilane [NH;C;], cyano
nitrile silane [CN],
trimethylsilane [C~], sulfoxyl propyl silane [S04C;], dimethylsilane [C,],
acidic cation-
7
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
exchange coating [SCX]. basic quaternary ammonium anion exchange coating
[SAX].
dihydroxypropyl silane [diol]), into a composite particle 0.1 - 10.0 um in
diameter. By wav
of example, the following silicas are useful for forming the composites of the
invention.
Eka Nobel Kromasil~
PaclaneParticlePore Pore SurfaceCarbonPhase Bonded End
lYlaterialShape Size Volume Area Load Type Phase Cap
& Size (t~,) (ml/~) (m''/g)(%) Coverage
(um) (umol/m'')
Kromasil5,x,7,10,100 0.9 340 - (elemental
Silica 13.16 analysis)
KromasilS,~,7,10,100 0.9 340 =l.7 Vlonomeric:1.3 -
C1 13,16
KromasilS,5,7,10,100 0.9 340 3 ivlonomeric3.7 ~'es
C4 13,16
KromasilS,~,7,10,100 0.9 340 12 iVlonomeric3.6 ~c'es
C8 13,16
KromasilS,5,7,10,100 0.9 340 19 iVlonomeric3.2 ~'es
C18 13,16
8
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WO 01/28587 PCT/US00/28615
EiYI Science
Paekine ParticleFore Fore Surface Carbor.~ PhaseBonded F,;,'
Material Shape Size Volume Area Load Type Phase Cap
& Size(~) (mllg) (m'/e) (%) Coverage
(gym) (~mol/m')
Lichrosorb
Si
60 I, 60 - 660 0 - - No
5,
10
Lichrosorb
Si
100 I, 100 - 420 0 - - No
~,
10
Lichrosorb
10 60 - 160 16.0 vlonomeric 1.66 No
5
I
RP-18 ,
,
Lichrosorb
g 10 60 - - 9.0 >vl~omcric 0.78 Vo
~
I
Rp- ,
,
LichrosorbI, 60 O. 560 1. - . 2.= Yes
6, l
10
RP-select
B
LichrospherS, 60 0.95 660 0 - 0 No
Si 3,
~,
60 10
.
LichrospherS, 100 1?6 420 0 - 0 No
Si 5,10
100
LichrospherS, 60/1001?6 360 12.6 - -l~l ~o
3,
6,
RP-8 10
LichrospherS, 60/1001.25 350 t3 - s.2 ~'es
RP-8 FJC 3,5,10
LichrospherS, 100 1?6 360 '-lv - ''9 Vo
3,
6,
RP-18 10
LichrospherS, 100 1.26 360 21.6 - - ~'es
3,
S,
RP-18 FJC 10
LichrospherS,3,6,100 I.26 350 -
CN 10
LichrospherS, 100 1.25 350 4.5 - 3.8 -
3,
5,
NH2 10
LichrospherS, 100 1.25 350 8.3 - 4.0 -
3,
~,
Diol 10
LichrospherS, 60 0.9 360 12.0 - ~.2 Yes
3,
5,
RP-select 10
B
9
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/2861~
Packing ParticlePore Pore Surface Carbonrnase tsonaea ~.na
MaterialShape Size Volume Area Load Type Phase Cap
& Size (~) (ml/g) (m''/g) (%) Coverage
(umolim-)
( um)
InertsilS, 5 150 - 320 0 - -
Silica
InertsilS, 5 150 - 320 13..5 iVlonomeric3.23 Yes
ODS-2
InercsilS, 3, 100 - 450 15 Monomeric - -
5
ODS-3
InertsilS, 5 150 - 320 10.5 iVionomeric.:.? ~'es
C8 i
InertsilS,5 100 - 450 10 Monomerie - 1'es
C8-3
InertsilS, 5 150 - 320 10 Nlonomeric2. i ~ es
Ph 7
(Phenyl)
InertsilS, 5 100 - 450 10 Monomeric - ~'es
Ph-3
(Phenyl)
InertsilS, 5 150 - 320 7.5 Monomeric 3. i7 Yes
C4
InertsilS, 5 80 - 450 16 Monomeric - ~'es
80~.
InertsilS, 10 100 - 350 14 - -
Prep
ODS,
CB,Si
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
Vvdac/The Separations Group
PackincParticlePore Pore SurfaceCarbon Phase Bonded End
MaterialShape Size Volume Area Load Type Phase Cap
& Size (~) (ml/e) (m'/g)(%) Coverage
-
(gym)
(umol/m
)
Vydac SD, 300 0.6 90 8 Polymeric4.16 Yes
5,
201TP 10
C18
Vydac SD, 300 0.6 90 8 Polyermic4.16 Yes
~,
218TP 10
C18
Vydac SD, 300 0.6 90 3 Polymeric=t.89 Yes
p,
214TP 10
C4
Vydac S, ~, 80 0.8 4~0 13.~ - 1.~3 -
201HS
C18
5
CA 02387925 2002-04-18
WO 01/28587 PCT°/US00/28615
Waters
Packing ParnclePore Pore SurfaceCarbon Phase Bonded End
Material Shape Size Volume Area Load Type Phase Cap
& Size (~) (ml/g) (m''/g)(%) Coverage
(pm) ( umol/m-)
uBondapakI, 10 12~ 1.0 330 10 Monomeric1.46 ~-es
C18
~BondapakI, 10 12~ I.0 330 8 - 2.08 Yes
Phenyl
pBondapakL 10 125 1.0 330 3.~ - 1.91 No
NH2
~BondapakI.10 12~ 1.0 330 6 - 2.86 Yes
CN
pPorasil L 10 125 1.0 330 - - - No
Silica
Novapak S, 4 60 0.3 120 ~ - 3.=t 1 ~-es
C18
Novapak S, 4 60 0.3 120 ~ - 2.3=1 Yes
Phenyl
Novapak S, 4 60 0.3 I20 2 - 1.6~ Yes
CN
Novapak S, 4 60 0.3 120 0 - 0 No
Silica
Resolve S, ~, 90 0.~ 1 i5 10 - 2.76 Vo
C18 10
Resolve S, ~, 90 0.~ 17> j - 2.~8 No
C8 10
Resolve S, ~, 90 0.~ 17~ 3 - 2.~3 No
CN 10
Resolve S. ~, 90 0.~ 175 0 - 0 No
Silica 10
SpherisorbS, 3, 80 0.~ 220 0 - 0 No
>>
Silica 10
SpherisorbS, 3, 80 0.~ 220 7 Monomeric1.47 Partial
5,
ODS-1 10
12
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2002-04-18
WQ 01/28587 PCT/US00/28615
Pacicine FarticiePore Fore SurfaceCarbonPhase Bonded )=nd
Material Shape Size Volume area Load Type Phase Cap
& Size (P,j (ml/~) (mr/g)(%) Coverage
(gym) (p.molim~)
SpherisorbS, 3, 80 0.~ 220 12 iVlonomeric2.72 Yes
~,
ODS-2 10
SpherisorbS, 3, 80 0.~ 220 6 Monomeric 2.51 Yes
~,
C8 10
SpherisorbS, 3, 80 0.~ 220 6 Monomeric 2.27 ~'es
~,
C6 10
SpherisorbS, 3, SO 0.~ 220 3 ~tonometic1.08 Partial
~,
Phenyl 10
SpherisorbS, 3, 80 0.~ 220 3.~ Monomeric 2.3 7 No
~,
CN 10
SpherisorbS, 3, 80 0.~ 220 2 iVlonomeric1.~8 Vo
~, .
NH2 10
SpherisorbS, 5, 80 0.5 220 - - - Rio
SAX 10
SpherisorbS, ~, 80 0.5 220 - - - -
SCX 10
Symmetry S 100 - 340 19 - 3.09 Yes
13
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YMC
PackingParticle Pore Pore SurfaceCarbon Phase Bontied End
Ivlater~aShape Size Volume Area Load Type Phase Cap
I & Size (A) (ml/g) (m'/g)(%) Coveraee
(gym) (umol/m-)
C18-A S, 120 1.0 -300 17 ivlonomeric- ~'es
3,5,7,10,15
C18- S, 120 1.0 -300 17 Monomeric- ~'es
AM 3,5,7,10,15
ODS- S, 120 1.0 -300 16 Nlonomeric- ~-es
AQ 3,5,7,10,15
C8 S, 120 1.0 -300 10 Monomeric- ~'es
3,5,7,10,15
Phenyl S, 120 1.0 300 9 Monomeric- Yes
3,5,7,10,15
C4 S, 120 1.0 -300 7 iVlonomeric- yes
3,5,7,10,15
Basic S, - - - - Monomeric- yes
3,5,7,10,15
Note: Bonded phase coverage calculated as per Sander, L.C., and Wise, S.A.,
,anal. Chem., ~6: ~04-
510, 1984. Material characteristics obtained from literature published by the
material manufacturer or an
authorized representative thereof.
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The powders are mixed in a planetary ball, or attrition mill in the presence
of a iiquid.
for example, ethanol, to inhibit oxidation of the iron. The liquid may also
serve as a lubricant
during the milling of the iron and ceramic powder, to produce the appropriate
particle size
distribution. It also may reduce compacting of the ceramic during processing.
As a result,
the porosity of the ceramic deposits in the composition is maintained so as to
maximize
adsorption capacity of the particles.
The mixture is put into a standard laboratory planetary ball, or attrition
mill of the
type used in powder metallurgy. The mill holds canisters containing the iron
and ceramic
powders, ethanol, and metal or metal alloy balls of various diameters. For
example, the mill
can have 6 mm diameter balls composed of case hardened metal carbide. An
appropriate
amount of a liquid (e.g., ethanol), is added for lubrication. Depending on the
twe used, the
mill is run between 2 and 1=1 hours at speeds of 100 rpm to 1000 rpm. It is
believed that mill
speeds over 1000 rpm could create an undesirable quantity of overly small
particles.
Appropriate liquids and milling conditions are easily determined by any person
having
ordinary skill in the art.
After joint deformation of the iron-ceramic mixture, the particles are removed
from
the mill and separated from the grinding balls, for example, by a strainer.
The particles may
be re-suspended in ethanol and homogenized to separate the particles from one
another. The
ethanol is removed, for example, by rotary evaporation, followed by vacuum
drying. Any
suitable drying technique may be employed, for example, in a vacuum oven
(purging ~=).
Particles should be handled so as to protect against oxidation of the iron,
for example, in a
nitrogen environment.
The resulting dried powder may then be sieved or magnetically separated to
obtain
the desired fraction of product providing the desired magnetic susceptibility
and therapeutic
or diagnostic binding capacity. The product is then packaged into dosage units
in a nitrogen-
purged glove box and terminally sterilized. Any suitable sterilization
technique may be
employed. For example, the iron-ceramic particles may be sterilized using
gamma
irradiation and the aqueous solution of excipients may be sterilized by
autoclave.
When ready for use, the biologically active agent or agents are adsorbed to or
precipitated onto the composite. The composite, with the active agent
adsorbed, is
administered to the patient in a suspension of the composite in a sterile
diluent.
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The iron-ceramic particles are useful as a carrier for delivering one or more
adsorbed
biologically active substances to specific body sites under control of an
external magnetic
field. As used herein, the term "biologically active substance" includes
substances useful for
in vivo medical diagnosis and/or treatment.
Biologically active substances include, but are not limited to,
antineoplastics, blood
products, biological response modifiers, anti-fungals, antibiotics, hormones,
vitamins,
proteins, peptides, enzymes, dyes, anti-allergies, anti-coagulants,
circulatory agents,
metabolic potentiators, antituberculars, antivirals, antianginals, anti-
inflammatories,
antiprotozoans, antirheumatics, narcotics, opiates, diagnostic imaging agents,
cardiac
glycosides, neuromuscular blockers, sedatives, anesthetics. as well as
paramagnetic and
radioactive particles. Other biologically active substances may include, but
are not limited
to, monoclonal or other antibodies, natural or synthetic genetic material and
prodrugs.
As used herein, the term "genetic material" refers generally to nucleotides
and
polynucleotides, including nucleic acids, RNA and DNA of either natural or
synthetic origin,
including recombinant, sense and antisense RNA and DNA. Types of genetic
material may
include, for example, genes carried on expression vectors, such as plasmids,
phagemids,
cosmids, yeast artificial chromosomes, and defective (helper) viruses,
antisense nucleic acids.
both single and double stranded R1~IA and DNA and analogs thereof. Also
included are
proteins, peptides and other molecules formed by the expression of genetic
material.
For in vivo diagnostic imaging, the type of detection instrument available is
a maior
factor in selecting a given radioisotope. The radioisotope chosen must have a
type of decay
that is detectable for a given type of instrument. Generally, Gamma radiation
is required.
Still another important factor in selecting a radioisotope is that the half
life be long enough
so that it is still detectable at the time of maximum uptake by the target,
but short enough so
that deleterious radiation with respect to the host is minimized. Selection of
an appropriate
radioisotope would be readily apparent to one having average skill in the an.
Radioisotopes
which may be employed include, but are not limited t0 99mTc, !~'Pr,'6!Tb,
!s6Re, and !ssRe.
Additionally, typical examples of other diagnostically useful compounds are
metallic ions
including, but not limited to !!!In, 9'Ru,6'Ga, 6gGa,''-As, s9Zr,
and'°!TI. Furthermore,
paramagnetic elements that are particularly useful in magnetic resonance
imaging and
electron spin resonance techniques include, but are not limited to !'~Gd, "Mn,
!6''Dy,''Cr,
and 5~'Fe.
16
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
It is also noted That radioisotopes are also useful in radiation therapy
techniques.
Generally, alpha and beta radiation is considered useful for therapy. Examples
of therapeutic
compounds include, but are not limited to'''P, ~s6Re, ~sBRe, ~'3I, ~'''I,
'~°Y, m6Ho, ~"Sm, ~~'ZPr,
i:~3Pr o9Tb ~6i.Lb mIn ~;Br '-~'-Bi '-oBi '-'eRa z~oPo n_Pt o:mPt '-s'Fm n~Dy
~o~Pd
> > > > > > > > > > > > > ,
~'~Sn, ~''Te, and 21 ~At. The radioisotope generally exists as a radical
within a salt, however
some tumors and the thyroid may take up iodine directly. The useful diagnostic
and
therapeutic radioisotopes may be used alone or in combination.
The iron-ceramic composite particle surpasses previous inventions by utilizing
metallic iron that has a higher magnetic susceptibility than iron oxides that
facilitates and
expedites mobility to the treatment site. Advantages over current iron-carbon
composite
products include surface binding versatility, as well as biocompatibilitv and
biodegradation
properties of ceramics that are relatively well known.
As a general principle, the amount of any aqueous soluble biologically active
substance adsorbed can be increased by increasing the proportion of ceramic in
the particles
1~ up to a maximum of about ~0% by mass of the composite particles without
loss of utility of
the particles in the therapeutic treatment regimens described in this
application. In many
cases it has been observed that an increase in the amount of adsorbed
biologically active
substance is approximately linear with the increase in ceramic content.
However, as ceramic
content increases, the susceptibility, or responsiveness, of composite
particles to a magnetic
field decreases, and thus conditions for their control in the body worsen
(although adsorption
capacity increases). Therefore, it is necessary to achieve a balance in the
iron:ceramic ratio
to obtain improved therapeutic or diagnostic results. To increase the amount
of agent given
during a treatment regimen, a lamer dose of panicles can be administered to
the patient, but
the particles cannot be made more magnetic by increasing the dose. Appropriate
ratios may
2~ be determined by any person having average skill in the art.
It has been determined that the useful range of iron:ceramic ratio for
particles
intended for use in in vivo therapeutic treatments as described in the
application is, as a
general rule, from about 99:1 to about 5:9~ for example about 80:20 to about
60:40. The
maximum amount of the biologically active substance that can be adsorbed in
the composite
iron:ceramic carrier particles of any given concentration of ceramic will also
differ
depending upon the chemical nature of the biologically active substance, and,
in some cases,
17
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/2861~
the type of ceramic used in the composition. Any person having ordinary skill
in the art will
be able to determine the proper ratio for the desired application.
Because it is convenient to prepare and market the carrier particles in a dry
form, the
excipients may be prepared in dry form,and one or more dry excipients are
packaged together
with a unit dose of the carrier particles. A wide variety of excipients may be
used, for
example, to enhance adsorption or desorption, or to increase solubility. The
type and amount
of appropriate dry excipients will be determined by one of skill in the art
depending upon the
chemical properties of the biologically active substance. Most preferably, the
package or kit
containing both the dry excipients and dry carrier particles is formulated to
be mixed with the
contents of a vial containing a unit dose of the drug and sufficient amount of
a biologically
compatible aqueous solution, such as saline, as recommended by the drub
manufacturer, to
bring the drug to a pharmaceutically desirable concentration. Upon mixture of
the solution
containing the dilute drug with the contents of the kit including the dry
components (i.e., the
dry carrier particles and dry excipients), the drug is allowed to adsorb to
the carrier panicles.
forming a magnetically controllable composition containing a therapeutic
amount of the
biologically active substance adsorbed to the carrier panicles that is
suitable for in vivo
therapeutic or diagnostic use.
Alternatively, a liquid kit may be employed. Here, the carrier particles are
contained
as one unit, for example, in a vial, while the aforementioned excipients are
contained in
another unit in the form of an aqueous solution. At the time of
administration. the
ferroceramic particles are mixed with the contents of a vial containing a unit
dose of the drug
and sufficient amount of a biologically compatible aqueous solution, such as
saline, as
recommended by the drug manufacturer, to bring the drug to a pharmaceutically
desirable
concentration. Subsequently, the resulting particles having the biologically
active substance
adsorbed thereon, are mixed with yet another unit containing the excipients in
aqueous
solution. Any suitable sterilization technique may be employed. For example,
the
ferroceramic particles may be sterilized using gamma irradiation and the
aqueous solution of
excipients may be sterilized by autoclave. Use of autoclave undesirably
oxidizes the
ferroceramic particles.
Also, when the biologically active went to be adsorbed to or deposited onto
the
microparticles is soluble in an aqueous medium, the buffer used can have an
impact on the
18
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
overall binding. Any person having ordinary skill in the art would be able to
determine the
most appropriate buffer.
A diagnostic or therapeutic amount of biologically active substance adsorbed
to the
carrier particles will be determined by one skilled in the art as that amount
necessary to effect
diagnosis or treatment of a particular disease or condition, taking into
account a variety of
factors such as the patient's weight, age, and general health, the diagnostic
or therapeutic
properties of the drug, and the nature and severity of the disease.
A number of considerations are involved in determining the size of carrier
particles to
be used for any specific therapeutic situation. The choice of particle size is
determined in
part by technological constraints inherent in producing the particles under
0.2 ,um in size. In
addition, for particles Less than about 1.0 ~.m in size, the magnetic control
in blood flow and
the carrying capacity is reduced. Relatively lame particle sizes can tend to
cause desirable or
undesirable embolization of blood vessels during injection either mechanically
or by
facilitating clot formation by physiological mechanisms. The dispersion may
coagulate,.
which makes injections more difficult, and the rate at which biologically
active substances
desorb from the particles in the targeted pathological zones may decrease. The
method (such
as is described below) of milling together a mixture of iron and ceramic
powders produces an
irregularly shaped form with a granular surface for the particles, and results
in a particle
population having an average major dimension of about 0.1 ~cm to about ~.0
~cm.
?0 Because the iron in the particles described in this invention is not in the
form of an
iron oxide, as is the case in certain previously disclosed magnetically
controlled dispersions,
the magnetic susceptibility, or responsiveness, of ferroceramic particles is n-
~aintained at a
high level.
The iron:ceramic particles are characterized by particles of iron and
particles of
ceramics bound together. The two components are maintained as individual
entities. The
characteristic substructure of the particles formed during the process of
joint deformation of
the mechanical mixture of iron and ceramic powders, also increases the
magnetic
susceptibility of iron inclusions in ferroceramic particles as compared with
iron particles
having other types of substructure.
Because of the large surface of ceramic deposits in the particles, the
adsorbed
biologically active substance can comprise about 100% - ~0°,% by
weight, relative to the
ceramic fraction of the particle, that being variable from about ~% to 9~% of
the initial
19
CA 02387925 2002-04-18
W~ 01/28587 PCT/US00/286I5
particle mass, and most preferably from 15% to 60%. In different terms, this
can be up to
about 200 mg of adsorbed biologically active substance per gram of panicles.
Therefore, in
use, much less of the carrier is injected to achieve a given dose of the
biologically active
substance or, alternatively, a higher dosage of the biologically active
substance per injection
is obtained than is the case with some previously known carriers.
The following describes a method for producing small quantities of the
ferroceramic
composition of this invention, it being understood that other means and
mechanisms besides
milling could be conceived of for jointly deforming iron and ceramic powders,
which
comprise the essential starting elements for production of the carrier. The
procedure utilized
exerts mechanical pressure on a mixture of ceramic and iron particles to
deform the iron
particles and develop a substantial substructure, which captures the ceramic.
The formation
of the ferroceramic particles is accomplished without the addition of heat in
the process
(although the mixture heats up during the mechanical deformation step), and is
conducted in
the presence of a liquid, for example ethanol, to inhibit oxidation of the
iron and to assure
1~ that the particles produced are clean (sterile). The liquid may also serve
as a lubricant during
the milling of the iron and ceramic powder, and may reduce compacting of
ceramic during
processing. As a result, the density of the ceramic deposits in the
composition is maintained
so as to maximize adsorption capacity of the particles.
As the joint deformation of the particles and ceramics continues, there
develops at the
interface of the two solids a third phase comprised of a molecular mixture of
iron and
ceramic. This interface stabilizes the particle such that it is durable to
sterilization and in
vivo use. This interface is expected to form with other types of ferro
particles, such as
ferrocarbon, as molecular mixtures of iron and carbon exist in nature or can
be fotzned by
smelting, for example, cementite and steel. Ferroceramic mixtures are not
commonly known
or manufactured such that a molecular mixture may be found at the interface of
the two
substances.
For example, to produce panicles having an average of about 7~:2~ iron:ceramic
ratio
by mass, one part of substantialiv pure iron particles having average
diameters from 0.1 :gym
to 5 f.em in size are mixed with about 0.1 to 1.0 parts by weight of
substantially pure ceramic
granules (typically about 0.1 ~cm to ~.0 ,um in diameter). The iron particles
and ceramic
granules are mixed vigorously to achieve good distribution throughout the
volume. Each
biologically active substance should be evaluated individually with the
various types of
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
ceramics in order to determine the optimum reversible ceramic binding. Factors
such as pH,
temperature, particulate size, salts, solution viscosity and other potentially
competing
chemicals in solution can influence adsorption capacity, rate, and desorption
parameters.
The mixture is put into a standard laboratory planetary ball, or attrition
mill of the
type used in powder metallurgy. For example, the mill can have 6 mm diameter
balls. An
appropriate amount of a liquid, for example ethanol, is added for lubrication.
The mixture is
milled for between l and 12 hours, or for the time necessary to produce the
panicles
heretofore described. Depending on the mill used, the speed of the mill may be
anywhere in
the range from about 100 rpm to about 1000 rpm (typically about 300 rpm.
After joint deformation of the iron:ceramic mixture, the particles are removed
from
the mill and separated from the Grinding balls, for example, by a strainer.
The particles may
be resuspended in ethanol and homogenized to separate the particles from each
other. The
ethanol is removed, for example, by rotary evaporation, followed by vacuum
drying. Any
suitable drying technique may be employed. Particles should be handled so as
to protect
against oxidation of the iron, for example, in a nitrogen envtrontnent.
After drying, the particles should be collected according to appropriate size.
For
example, the particles may be passed through a 20 ~m sieve and collected in an
air cyclone
to remove particles larger than 20 Vim. The cyclone only collects particles of
a certain size
and density, providing a method for removing fines and loose ceramic. The
sieved particles
may be packaged under nitrogen and stored at room temperature.
Particles may be subaliquoted into dosage units, for example, between 50 and
500 ma
per dose, and may be further overlayed with nitrogen, for example. Dosage
units may be
sealed, for example, with butyl rubber stoppers and aluminum crimps. Dosage
units may
then be sterilized by appropriate sterilization techniques, for example, gamma
irradiation
between 2.5 and 4.0 Mrads. Other sterilization techniques may also be used,
for example, dry
heat and electrobeam sterilization.
When ready for use, or before packaging if the car-ier is to be prepared with
a
preselected biologically active substance already adsorbed thereon, about 50
mG to 150 mg
(about 75 mg to about 100 mG is preferred to be absolutely assured of maximum
adsorption)
of the biologically active substance in solution is added to 1 Gram of the
carrier. When ready
for application to a patient, the combination is placed into suspension (for
example, in 5 to 10
ml) of a biologically compatible liquid such as water or saline utilizing
normal procedures.
21
CA 02387925 2002-04-18
W~ 01/28587 PCT/US00/28615
Example 1
A composite particle composed of silica gel and iron was manufactured and
preliminary characterization was performed. Characterization included particle
sizing
analysis (light scattering technique), surface area, pore size analysis,
scanning electron
microscopy and doxorubicin binding. Tests show that 9~% of the final product
has particles
that are less than 1.11 m and have a mean (volume) diameter of 0.92 m. Results
from
surface area analysis show the iron-silica gel composite to have a total
surface area of 48
m2/g and a total pore volume of 0.19 cc/g. SEM pictures reveal discrete
particles made of
both iron and silica gel components (Figures 1 and 2). Preliminary doxorubicin
binding
assays (Figure 4) show correlation between the concentration of bound (Q) and
unbound (C)
doxorubicin.
Doxorubicin Binding for Iron-Silica Gel
Composite
120
~ ~ 100
v ~ 80
~ 60 ~ ,
0 ~ ~ ~ i
a
0 20 ~-
0'
0 200 400 600 800 1000
Cunbound (p.9 ~O~ml solution)
22
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
Example 2
A composite particle composed of silica-C18 and iron was manufactured and
preliminary characterization was performed. Characterization included particle
sizing
analysis (light scattering technique) and doxorubicin binding. Tests show that
95% of the
final product has particles that are iess than 1.60 m and have a mean (volume)
diameter of
1.~8 m. Preliminary doxorubicin binding assays (Figure 5) show a linear
correlation
between the concentration of bound (Q) and unbound (C) doxorubicin.
Doxorubicin Binding on Iron-Silica C18
Coin pos ite
60
t 50 v
,
v 40
w;
E ~ ,
I ~ 30
~ 0
o
~ 20
m
CJ ~ 10
~
0
0
200
400
600
800
1000
C
(Unbound)
[ug
DOXImI
solution)
23
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
Example 3
In order to bind a biologically active substance for targeted delivery,
initially, the
structure of the agent would be evaluated. Paclitaxel, for example, contains
three -OH
groups and three benzene rings. Using the information contained in Table l,
binding would
be attempted using those derivatives for benzene rings and -OH groups. First-
line silica
derivatives would include bare silica, C8 and C 18. Second-line derivatives
would include
phenyl, C1, C2, C4 and C6. Additional silica derivatives would be tested based
on the results
from experiments. The derivatives should be easily determinable by any person
having
ordinary skill in the art. Neoplastic agents may be especially useful with the
particles of the
invention. Examples of other useful neoplastic agents are exemplified in Table
2.
Table 1: Examples of Functional Characteristics of Agents and Silica
Derivatives
Functional CharacteristicsPotential Silica Derivatives for Binding
.
-OH groups Bare silica
Open chain structure Bare silica
Benzene rings C8, C 18
Long alkanes C8, C18
Positive char2e Cation exchange (i.e. sulfoxyl (SO,),
carboxyl . SCX)
Negative charge Anion exchange (i.e. Quartinary (S AX),
diethylaminoethyl)
Mixture of rings and -OH Phenyl, C 1, C2, C4, C6
groups
24
CA 02387925 2002-04-18
WO O1/2858'~ PCT/US00/28615
Table 2: Agents Useful in Neoplastic Disease
Agent ! :gent dame ! Trade Name Abbr. j
NmH~;eN Vlachlorathamine
~ ALKYL.4TING alua-r~ICU, ~ H~;=
AGENTS Vtustaroen,
nitrogen
mustard
Cyclophosphamide Cvtoxan, CTS j
Endoxan
Ifosfamide Ifex IFS
Phenylalanine mustardMelphalan, L-P.4VI
Alkeran
Chlorambucil Lukeran
CLR
ETIIYLENIMINF:I TriethylenethiophospheramideI T-TEP.~
Thiotepa j
oeRIVnTIVES ,
.1LKYLSDLFONATE1~ Busulfan ~ ~ MYL
Myleran
NITR()1UURIi.ISI Cyclohexyl-choiorethylLomusnne. CCNU I
CEENL~
nitrosourea ~
I, 3 bis-[2-chloroethyl)-Carmustine. BCNL.I
BiCNU
i nitrosourea
~
Streptozotocin Zanosar STZC
Semustine
TRL1ZENES ~ Dimethyl ttiazenoDacarbazine DTIC
imidazole
I carboxamide ~ ~
ANTIMETABOLITES FULIC.~CID:\N,\L~)~:1~ Methotraxate ~ pterin ~ VITX j
Ametho
f~YRIrtIDINE:1.V.V.Wp j-fluoro-2-deoxyuridine! FUDR
:, Floxuridine
~-fluorouracil ~ ~-FU
Cytarabine,
Cvtosar
Cytosine arabinoside ARA-C
PURINEnNnuna6-Mercaptopurine ~ 6-VP
Purinethol
6-Thioeuanine j 6-TG
Tnioeuanine
Deoxycoformycin ~ VW-?6
Pentostatin
vI Nc.v
NATUR4L OR SEMI- .wvu n Vinblastine ~'elban VLB
D
SYNTHETIC PRODUCTS ~ ~ VCR t
Vincristine Oncovin
.~NITfiIUTICSDoxorubicin ~ Adtiamycin ADR '
'
Mitoxantrone Novantrone NDV
i
~
Baunorubicin Daunomycin DNR
Bleomycin ~ Blenoxane BLEO
Dactinomycin Actinomycin - - - i
D,
(
I Cosmegen
~
Mithramycin I~tithracin
Mitomycin C ~ Mutamvcin ~(ITO-C.
~
I I MVC
T.VL\NES Paclitaxel I Taxol TXL
I ~
- e,IZYMES L-aspara_ainase Elspar L-ASP
~ I ~ i
EPIPODUPFIYI~TOXINSEtoposide I ~'epesid VP-15
Teniposide ~ ~ vV(_~6 I
~'umon
,
MISCELLANEOUS PLATINUM Cis-diamminedichloro-Cisplatin, CDDP
I ~ Plannol
CnORDINATIDN
cnMPLExES platinum II
~
Carboulatin ~ Paraplatin CBp
SUBSTITUTEDHydroxyurea Hydrea HXU
UREr~ ~ ~
HETHYLHYDR.1ZINEProcarbazine Matulane PROC
DERIVt~TIVE~ I
I
A(:RIDINE Amsacrine Emcyt m-A,~j
DERIVATIVE -
'
A
msi I
dyl
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
Agent ~ Agent Name ~ Trade Name ; Abbr.
_ Ai _ ~ esTKO(:e,ISDiethylstilbestrol DES
HORMONE INHIBITORS Conjugated EstrogensPrematin
Ethinyl EstradiolEstinvl
ANDRD(:ENS Testosterone propionate- - - TES
Fluoxymesterone Halotestin,
Ora-
Testryl, Utandran
m-nyaroxyprogesteroneuelalutm
caproate
MedroxyprogesteroneProvera
acetate
Meeestrol acetateMeeace
LEUPii()LIDE I LUprOn
Goserelin acetateI Zoladex
t'reaniSOne j
Tamoxifen . ~iolvadex i
Aminoglutethimide ~ Elipten, Cytadren
Flutamide ~ Eulexin
26
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
Example
The adsorption capacities of hydroxyapatite panicles and the iron-
hydroxyapatite
composite particles were determined by a doxorubicin binding assay. The
Langmuir
adsorption isotherms were determined from doxorubicin binding data at several
concentrations and the total drug loading capacities were calculated from the
inverse of the
slope of the isotherms. Figure 12 shows the isotherm for the iron-
hydroxyapatite composite
particles, which had a total capacity of 33 micrograms doxorubicin per
milligram particles.
Figure 13 shows the isotherm for the hydroxyapatite alone, which has a binding
capacity of
~3 micrograms doxorubicin per milligram particles. The difference in the drug
binding
capacity between the hydroxyapatite and the iron-hydroxyapatite composite
material is due
to the difference in compositions of these samples: the composite material of
this example
has ~ 2~% per weight of hydroxyapatite.
tangmui~ Isotherm
~-_0 . ~ = L~J o..~t ~T~t"i -
r~' = 0.9925
1q I
>6
i 1
t2
a 1
a $.
2 ..
0.0 2~0 ~~ :0C 0 oCO.C 8~~.7 t000
1) r~ r~~nlrn~l
7'7
CA 02387925 2002-04-18
WO O1/2858~ PCT/IJS00/28615
Example J
Iron-hydroxyapatite composite particles were loaded with doxorubicin by
soaking the
particles in a concentrated aqueous solution of the drug. The desorption
profile was
determined in a semi-dynamic assay by measuring the amount of doxorubicin
released from
the particles incubated in aliquots of human plasma at 37°C. Figure 14
shows that the drug is
effectively released from the microparticles as a function of time.
Amount
DatorLed
~.
n,7,e
my n,
,sss
uao.o _._. .. .. _.. .... ___ . ..
.. _ ..._.___.._. _ . .
_ .
G G
G g g g
G g
0
. g
a ,~.a
~
a
i
i 1
t i i
_,
1 mrc R..o,a si.~.a
m.r .e~av,n
500.0 r I a ITC NB070.51
~
i 1 ~ wTC Rfi6Qw
i
C AfTC 1170 (Nrtr
Rnlmnn SOtnW
N Ola,
eah M ilAOmrC
~ - en 4vwmow a7,
j
___ .._....
._. ... ..,...,.._.......__.._ _~ ...._..-_._.__
0.0 20 r0 60 _..,.-__._......
0 00 100 t20 ,10 '
,60
TlrtHt
Iminutr~)
28
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
Example 6
Iron-hydroxyapatite micro particles were incubated with Indium-111 in PBS for
30min at 37° C and 1400rpm. The labeling efficiency was determined by
comparing the
amount of radioactivity in the incubation with the bound radioactivity after
two washes with
6 PBS. The inset in Figure 15 shows the resulting labeling efficiencies, which
were
approximately 60% after the second wash. The stability of the labeled
particles was tested in
both PBS and human plasma at 37°C. For each time point, the total
activity of the sample
was compared with the activity in the supernatant, After 12 days, the iron-
hydroxyapatite
micro particles in PBS retained more than 95% of the Indium-l l l and the
stability in of the
particles in plasma was about 90%. These results demonstrated that the
microparticles are
easily labeled with Indium canon and that the labeling is very stable in human
plasma.
100 .~ , .
9s .-.
~ _~.~_~~a___ ' __,_._...-
__.!_._.
as
"' 75 ' _
._ . _.,
> 70 ,._._,__.._.-_ .__._....,_._ _ . . ' . .........
m 65 -,._
_ I ~1~E._.
"'in'' Labeling in PBS pH 7.4
40 ~ HMTC-PBS
i -r- f-ffMC - PI2sm2
' ...,
0 24 48 72 96 120 144 168 192 216 240 264 288 312
TIfTte (hl
29
CA 02387925 2002-04-18
WO 01/28587 PCT/US00/28615
Example
The previous experiment was repeated using an Indium complex instead of the
Indium salt. Indium-111-oxyquinoline complex was used in the incubation step
after being
prepared by well know methods. The efficiency and stability were determined as
described
previously and the results are shown in Figure 16. The labeling efficiency
increased to over
90% after the second wash. The stability of the Indium-oxyquinoline labeled
micro particles
is very similar to the direct labeling, with more than 95% of the
radioactivity remaining
bound after 12 days in PBS and about 90% of the radioactivity still bound
after 12 days in
plasma. Thus, Indium complex can also be directly labeled in a very stable
manner onto the
particles.
100 ~ ~ . ~ , _. ,
w ~~ ~---
-.. - -
~ _..__-.. ~ _.-. _-.._.-t
0 80 .v ;
7g ,.__~_._.~_,_~____.;...._.-____-,_...._.._..._..__ _._.,
'> 70 _
0 65 401-SE
",~n-0~dne in PBS pH 7.4
c z .t_
-~ 4B - PBS
,.
40 'I ~ HMTC - PBS
35 ~ -r- i-fTMC - Plasma ~ ...., ._,
0 24 48 72 96 120 144 168 192 216 240 264 288
Time [h]
30
