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Description  |
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The present invention relates to drug delivery systems and more
particularly to a system for assisting in the delivery of a drug or
radiodiagnostic agent to a desired location within the animal or human
body.
Colloidal particles in the form of microspheres, microcapsules, emulsions
and liposomes, have been proposed as a means of directing drugs contained
therein to specific sites in the body. This concept, also known as drug
targeting, has been well described in a number of publications, review
articles and books. (see for example Davis, Illum, Tomlinson and McVie,
(editors) Microspheres and Drug Therapy, Elsevier, Amsterdam, 1984).
Colloidal carriers have been shown to perform well in in vitro tests but
their utility in vivo has been disappointing. It is known to be a
relatively simple matter to direct particles to the lung or to the liver
by exploitation of physical factors such as particle size. However, the
rapid and efficient capture of injected particles by the cells of the
reticuloendothelial system residing in the liver (namely the Kupffer
cells) does present a major obstacle to targeting colloidal particles
elsewhere. Indeed, in a recent review article by Poste and Kirsch
(Biotechnology 1: 869, 1984) and Posnansky and Juliano (Pharmacol. Revs.
36, 277, 1984) this very point was emphasised. Similarly, at a meeting of
the New York Academy of Science held in March 1984 (published in
Proceedings of the New York Academy of Sciences, Vol. 446, Editors
Tirrell, D. A., Donaruma, L. G. and Turek, A. B., 1985), on the topic of
polymers for drug delivery, many of the presenters of papers concluded
that it would be almost impossible to direct colloidal particles to other
sites than the liver and spleen when administration was by the
intravaneous route. The present invention provides a method whereby it is
possible to direct particles away from the reticuloendothelial system
residing in the liver and spleen by the use of surface coatings (and
surface grafting techniques).
Model particles for use in studying the fate of drug carriers are often
used in order to determine the scientific basis of drug targeting.
Polystyrene microspheres of different sizes have been particularly useful
in this respect. The small polystyrene particles of a size less than 100
nm are administered intravenously. They are taken up rapidly and
efficiently in the liver as measured by the non-invasive technique of
gamma scintigraphy or by studies on animals where organs are removed and
radioactivity levels are determined in such organs. Typically, more than
90% of the injected dose is found within the liver in a period of about 3
minutes (Illum, Davis, Wilson, Frier, Hardy and Thomas, Intern. J.
Pharmaceutics 12 135 (1982)).
It is an object of the present invention to provide a drug delivery system
which obviates the above problem and prevents such a rapid take up of an
injected dose by the liver.
According to the present invention there is provided a drug delivery system
comprising a number of particles containing an active drug, or a
diagnostic agent to include radioactive materials. The particles could be
for example, emulsions, microspheres made from natural and synthetic
polymers, or phospholipid vesicles, each particle being coated with a
material to form a composite particle which substantially prevents the
take up of the composite particle by the liver.
Preferably the particles are coated with a material that provides them with
both a hydrophilic coat that will minimize the uptake of blood components
and a steric barrier to particle-cell interaction. It is then found that
the amount being taken up by the liver is greatly reduced. One preferred
material is the block copolymer known as tetronic 908. This is a non-ionic
surfactant which is obtained by polycondensation of propylene oxide and
ethylene oxide on ethylenediamine. This coating material allows
intravenously injected particles to remain within the systemic circulation
with minimal uptake in the liver and spleen. Another preferred material is
the block copolymer known as poloxamer 407, a mixture of polyoxyethylene
and polyoxypropylene domains. This material also is effective at
preventing uptake of coated particles in the liver and spleen but directs
them almost exclusively to the bone marrow. Other members of the poloxamer
and poloxamine series have similar effects provided that the material
chosen has a sufficiently large hydrophilic domain for steric
stabilization. Typically an adsorbed layer thickness of about 100 Angstrom
or larger is required. This represents in the poloxamer series 60 or more
ethylene oxide units.
The mechanism of action of the materials resides in the structure of the
coating agent, namely that it has hydrophilic and hydrophobic domains. The
hydrophobic domain will anchor the coating to the particle surface and
prevent its displacement by plasma proteins. A suitable molecular weight
for this domain will be 4000-5000 Daltons. Hydrophobic domains include
polyoxypropylene groups as well as other hydrophobic moieties that can be
incorporated into polymer chains. For example, esterified maleic acid
groups.
The hydrophilic domain should be of a sufficient size and hydrophilic
nature to prevent (or at least minimise) the coating of the particle by
blood components (that is to minimise the phenomenon known as
opsonisation) as well as to provide a steric barrier so as to provide
steric stabilisation, a phenomenon well known in the field of colloid
science (Napper, Polymeric Stabilisation of Colloidal Dispersions,
Academic Press, London, 1983). Such steric stabilisation serves to prevent
the interaction of particles with the macrophage cells of the
reticuloendothelial system. A suitable molecular weight for the
hydrophilic domain is of the order of 5000-22,000 Daltons.
Embodiments of the present invention will now be described, by way of
example with reference to the accompanying drawings in which:
FIG. 1 shows the relationship of thickness of coating layer of polaxamers
and polaxamine on polystyrene particles;
FIGS. 2a and 2b show scintiscans of rabbits 3 hours after intravenous
administration of uncoated (a) and poloxamine 908 -coated (b) polystyrene
particles (60 nm);
FIG. 3 shows activity-time profiles for the uptake of uncoated and coated
(908) particles in the liver (n=3, means .+-.SEM);
FIG. 4 shows a graph of liver (spleen) uptake of fat emulsions labelled
with iodine-123;
FIG. 5 shows a graph of blood clearance of fat emulsions labelled with
iodine-123 (means values n=3, SEM not greater than 5%);
FIGS. 6a and 6b show gamma camera scintiscans of rabbits 3 hours after
intravenous administration of 131.sub.I -labelled polystyrene microspheres
(60 nm) (a) uncoated (b) poloxamer 407-coated;
FIG. 7 shows activity profiles for liver/spleen region after administration
of 131.sub.I -labelled polystrene microspheres " " uncoated, poloxamer
407 coated;
FIG. 8 shows a graph of uptake of poloxamer 407 coated microspheres in the
hind leg of the rabbit as measured by gamma scintigraphy;
FIG. 9 shows a graph of the activity in the circulating blood after the
administration of 131.sub.I -labelled polystyrene microspheres uncoated,
poloxamer 407 coated; and
FIG. 10 shows the distribution of 131.sub.I labelled polystyrene
microspheres in various organs 8 days following intravenous administration
uncoated microspheres poloxamer 407 coated microspheres.
Practical studies conducted in vitro with serum on the uptake of coated and
uncoated particles by mouse peritoneal macrophages have demonstrated the
importance of anchoring the polymer coating to the surface of the particle
and surface layer thickness.
Surface Layer Thickness
Polystyrene particles (60 nm in diameter) were dialysed against distilled
water for 3 days. 4.0% w/v aqueous solutions of the various poloxamers and
poloxamine were used to ensure that the final concentration, after
dilution to perform photon correlation spectrophotometer (PCS)
measurements, remained above the plateau level of the adsorption isotherm
i.e. above the critical micelle concentrations. Aliquots of 2.5% w/v
polystyrene particles and the coating solution were mixed and incubated at
room temperature overnight. The particle suspension was then diluted with
distilled water (20 .mu.l per 10.0 ml) and the pH adjusted with HCl or
NaOH. The thicknesses of the coating layers were then determined by
measuring the particle sizes for uncoated and coated particles of pH 2.1,
3.0, 5.5 and 9.5 using photon correlation spectroscopy.
Mouse Peritoneal Machrophage Studies
Polystyrene microspheres of 5.25 .mu.m in diameter were chosen for the
mouse peritoneal macrophage studies because uptake could be measured by a
microscopic method and van der Waals attractive forces would be a dominant
factor thereby allowing differentiation of the stabilising capacities of
different block copolymers. The polystyrene microspheres were dialysed
against distilled water for 3 days to remove any surfactant present. The
particles were then incubated for 24 hours with the different 2% w/v
poloxamer and poloxamine solutions. The concentrations of the coating
agent were chosen to ensure that at equilibrium the quantity of adsorbed
material was in the plateau region of the respective adsorption isotherms.
Female NMRI mice (Bommice, Monholtgaard Breeding and Research Centre Ltd.,
Ry, Denmark) weighing 20-25 g, were used to provide the peritoneal
macrophages. The animals were killed by cervical dislocation, the
peritoneal wall exposed and 5 ml of lavage medium (10 ml tissue culture
Medium E199 concentrate (10.times.) (Flow Laboratories), 10 ml swine
serum, 2.5 ml sodium bicarbonate 7.5%. 0.1 ml crystamycin, 6 mg heparin,
77.4 ml sterile water) injected into the peritoneal cavity followed by a
smaller volume of sterile air. The peritoneal wall was gently massaged and
the medium containing the macrophages was withdrawn and collected in a
sterile container kept on ice. The exudates from several animals were
routinely collected in this way and pooled. A cell count was conducted
using a Coulter Counter (model TAII). The viability of the macrophages was
tested by exclusion of tryptan blue and found to be in the order of 95%.
The macrophage suspension was adjusted to a final cell count of
1.0.times.10.sup.6 cells/ml and 1.25 ml of this suspension pipetted into
each 30 mm dish to give 1.25.times.10.sup.6 cells per plate. The plates
were incubated at 37.degree. C. in 95% air/5% CO.sub.2 for 3 h to permit
macrophage adherence to the bottom of the plate. After adherence the
medium was removed from the plates, the cells washed once with sterile
PBS, 1.25 ml of cell culture medium added (10 ml Medium E199 concentrate
(10 .times.), 10 ml Medium E199 concentrate (10 .times.), 10 ml swine
serum, 2.5 ml sodium bicarbonate, 0.1 ml crystamycin, 10 mg L-glutamine
and 79.9 ml sterile water), and the plates incubated at 37.degree. C. in
95% air/5% CO.sub.2 for 24 h. After incubation the medium was removed and
the cells washed once with sterile PBS. Then 2.5 ml cell culture medium
containing the appropriate number of coated or uncoated micropheres (5
particles per macrophage) was added to each plate and the plates incubated
in groups of 3 for 15, 30, 45, 60 and 90 min, as determined beforehand by
a time course experiment. Before counting the number of particles
phagocytosed by the macrophages, the media was removed from the plates,
the cells washed 2 times with sterile PBS and fixed with methanol for 5
min. Then the cells were stained with Giemsa (1:10) for 15 min and washed
with water. The plates were left to dry and the number of microspheres
phagocytosed by the macrophages was counted for a total of 100 macrophages
using a light microscope at a magnification of 500 times. The experiments
were performed in triplicate and results were expressed as the number of
microspheres phagocytosed by a 100 macrophages.
Experiments were also performed to determine whether free poloxamer and
poloxamine had any effect on the ability of the macrophages to phagocytose
particles. 2% w/w aqueous solutions of the coating agents were added to
the cells and left to incubate 1 hour. The solution was removed and the
cells washed 2 times with PBS. Then the cell culture medium containing the
uncoated microspheres was added and the degree of phagocytosis determined
as before. Free polymer was found not to influence phagocytosis and
therefore in all experiments the excess poloxamer of poloxamine was not
removed before incubation with macrophages.
The relative uptake of the various coated 5.25 .mu.m polystyrene particles
by mouse peritoneal macrophages and the relationship with surface layer
thickness are shown in Table 1 and FIG. 1. In general terms it can be seen
that the greater the adsorbed layer thickness the lower the relative
phagocytic uptake. These results are in line with the predictions of the
various theories put forward to explain the phenomenon of steric
stabilisation. Therefore, it appears that these theories can also be
applied to the interaction of particles with phagocytic cells.
Extrapolation of the regression line shown in FIG. 1 to zero phagocytic
uptake predicts that an adsorbed layer thickness of about 230 A would be
necessary to overcome van der Waals attractive forces between macrophages
and 5.25 .mu.m particles.
The size of the layer that would be sufficient to give the same stabilising
effect for much smaller (e.g. 60 nm) particles is difficult to predict
exactly. However, since the van der Waals attractive forces (VA) are
directly related to particle radius (a)
##EQU1##
where A is the composite Hamaker constant and h is the Planck's constant,
we would expect that an adsorbed layer thickness of about 100 A should be
adequate to provide not only steric stabilisation of 60 nm polystyrene
particles in terms of their aggregative propensity but also to a lack of
interaction with macrophages.
Embodiments of the present invention will now be described, by way of
examples:
EXAMPLE 1
The Organ Distribution and Circulation Time of Intravenously Injected
Colloidal Carriers Sterically Stabilized with a Block
Copolymer--Poloxamine 908
Methods
Polystyrene microspheres in the size range 50-60 nm were obtained from
Polyscience (Northampton, UK). The particle size was confirmed using
photon correlation spectroscopy. The particles were surface labelled with
Iodine-131 as described previously by L. ILLUM, S. S. DAVIS, C. G. WILSON,
N. W. THOMAS, M. FRIER and J. G. HARDY, Int. J. Pharm. 12, 135, 1982).
Poloxamine 908 (average Mw 25000: 80% average weight percentage of
polyoxyethylene chains) was obtained from Ugine Kuhlman Ltd., Bolton UK
and used as received.
Incubation of the polystyrene microspheres with a 2% w/v solution of
poloxamine 908 gave an adsorbed layer thickness of 134 A.
In vivo experiments were conducted with groups of New Zealand White rabbits
(3 kg) (n=3). Intravenous injections were given via the marginal ear vein
(polystyrene microspheres 0.3 ml, 4.times.10.sup.13 particles, 3 MBq
activity; emulsions 1.0 ml, 10.sup.12 particles, 3-4 MBq. Uncoated
polystyrene particles were administered in distilled water (control).
Particles coated with poloxamine 908 (24 hours equilibrium) were
administered either as the incubation mixture (containing 1% poloxamine
908) or in distilled water after the excess poloxamine had been separated
on a Sepharose CL4B column.
One group of rabbits was given similar repeated injections of poloxamine
coated polystyrene microspheres on five consecutive days. Another group
was given a dose of uncoated polystyrene microspheres 1 hour after the
injection of the coated material.
Blood samples were taken at suitable intervals and the activity counted in
a gamma counter. The distribution of the labelled particles in the liver
was followed by gamma scintigraphy. Dynamic and static images of the liver
distribution were analysed by creating regions of interest and compared to
whole body activity. The activity in the liver associated with the blood
pool was determined to be 25% of circulating activity using sequential
administration of Tc-99m labelled pyrophosphate (red blood cell label) and
Iodine-131 labelled microspheres (to provide a liver image). This value
agreed well with data for man and rat (see for example H. P. J. BENNETT
and C. McMARTIN, J. Endocr. 82, 33, 1979), J. W. TRIPLETT, T. L. HAYDEN,
L. K. McWHORTER, S. R. GAUTAM, E. E. KIM and D. W. A. BOURNE, J. Pharm.
Sci. 74, 1007, 1985).
Eight days after administration the rabbits were sacrificed and organs
removed. Total activity in selected sites and in the carcass was
determined using a large sample volume gamma counter.
Results
Uncoated polystyrene particles were taken up rapidly (t.sub.50% =55 s) and
efficiently (90% of dose in 2 min) by the liver and spleen while particles
coated with poloxamine 908 remained largely in the vascular compartment
and demonstrated little uptake in the liver/spleen region (FIGS. 2-3).
Similar results were obtained for coated particles separated from excess
poloxamine 908 using a sepharose CL4B column. Repeated injections of
polystyrene particles coated with poloxamine 908 (one injection per day
for 5 days) resulted in some uptake in the liver and spleen, but this was
largely associated with the blood pool in the liver. The injection of
uncoated polystyrene particles into rabbits 1 hour after they had received
a does of polystyrene particles coated with poloxamine 908 demonstrated
that the uncoated particles were mainly removed by the liver/spleen as for
untreated animals whereby demonstrating that the poloxamine 908 had caused
no impairment of the reticuloendothelial system.
The measurement of circulating levels of activity showed that the coated
particles remained largely in the vascular compartment while in
correspondence with the scintigraphic information, little of the uncoated
material could be found in the blood (Table 2). Interestingly, a
significant fraction of the administered does was not accounted for by the
blood level measurements. Scintigraphic measurements and organ level
determinations (see below) failed to reveal significant sites of uptake
(including bone marrow). Consequently, it is suggested that the coated
particles could be loosely associated with endothelial cells lining the
vasculature.
Levels of activity in the different organs eight days after injection are
shown in Table 3. The uncoated particles were found largely in the liver
and in the spleen while the coated particles were largely associated with
the carcass.
EXAMPLE 2
Intravenous Administration of Radiolabelled Emulsions and the Role of the
Block Copolymer--Poloxamine 908
This study was performed in order to establish whether the coating agent
poloxamine 908 would retain a biodegradable emulsion system solely within
the systemic circulation. Emulsions labelled with the gamma emitting agent
iodine-123, were injected intravenously into rabbits. Two control
formulations consisted of emulsions prepared using egg lecithin as the
emuslifier with and without added gelatin. The control system with gelatin
was chosen since it is well known that gelatin can have an important role
in directing colloidal particles to the liver; the process being mediated
by the adsorption of the blood component fibronectin. The role of the
different emulsifiers in controlling liver uptake as well as clearance
from the circulation was determined by scintigraphic imaging of the livers
of rabbits over a suitable period of time, as well as the removal of blood
samples and the counting of gamma activity. The oil chosen for this work
was soybean oil, the same component as used in the commercial product
Intralipid. Since this material is metabolised by the body, scintigraphic
and blood level data were collected over a period of 6 hours.
Methods
Animals
Female New Zealand White rabbits of an approximate weight of 2 kg were
chosen as the experimental model, 3 rabbits were chosen per group.
Preparation of emulsions
Soybean oil was labelled using the method of Lubran and Pearson (J. Clin.
Pathol. 11 (165) (1985).
Iodine-123 was chosen as the most suitable radio-nuclide from the
standpoint of its good imaging characteristics, its short half life and
its greater safety over iodine-131. The iodine-123 was obtained from
Harwell. The iodination method involves the covalent attachment of small
quantities of labelled iodine across the double bond of the unsaturated
components of the vegetable oil. This method has been used with success
previously and similar iodinated fatty acids have been used in the
radio-diagnostic field as myocardial imaging agents. The radio-labelled
oil was mixed with a further proportion of unlabelled oil and the mixed
oil was then emulsified with either poloxamine 908 (BASF) (2%) or with egg
lecithin (Lipoid) (1.2%). An ultrasonic probe system (10 min sonication)
(Dawe Soniprobe) was employed for this procedure. Previous investigations
using unlabelled oils has indicated that the particle size produced by
this method was of the order of 150 nm. This size is very similar to that
found in commercial fat emulsion products (e.g. Intralipid). One sample of
the egg lecithin stabilised emulsion was mixed with gelatin (2%) according
to the procedure described by Tonaki et al (Exp. Mol. Path. 25 189 (1976).
In this process some of the gelatin is adsorbed onto the surface of the
particles or may form a mixed emulsifying layer with the egg lecithin and
will thereby potentiate uptake of the emulsion in the liver, mediated by
adsorbed fibronectin.
Experimental procedure
The experimental animals were injected via the marginal ear vein using 1 ml
samples of the labelled emulsions. The oil content in the emulsions was
10%. The emulsions were followed by a 2 ml flush of normal saline.
Following injection the animals were placed on the measuring surface of a
gamma camera (Maxicamera, GEC. 40 cm field of view) tuned to the
photoenergy peaks of iodine-123. Dynamic images were taken every 15
seconds over a period of 15 minutes. Blood samples were removed from the
contralateral ear (0.5 ml). The scintigraphic images were stored on
computer and then analysed to provide information on the liver (spleen)
uptake. Blood samples were diluted and counted in a conventional gamma
counter. It is noted here that with gamma scintigraphy it is difficult to
distinguish between the liver and spleen in a live animal but, with
reference to FIG. 10 and to other results it is the liver which is the
dominant organ.
Results
Uptake of labelled emulsions in the liver and spleen region is shown in
FIG. 4. Mean values n=3, SEM not greater than=2%. Dotted line at 25%
indicates blood pool.
______________________________________
Values at 6 hours:
% uptake in liver
______________________________________
1.2% lecithin 34 .+-. 2
1% P-908 27 .+-. 2
1.2% lecithin + 0.3%
47 .+-. 1
gelatin
______________________________________
It can be seen that the extent of uptake is dependent upon the nature of
the emulsifier used in preparing the emulsions. Those prepared using
poloxamine 908 provided a liver uptake of approximately 25% while those
emulsified with egg lecithin had a value closer to 40%. The emulsions
containing the added gelatin had an uptake value of approximately 60%.
These liver uptake values for egg lecithin and P-908 systems are reflected
in the blood level versus time profile in that the emulsions stabilised by
egg lecithin demonstrate a much faster clearance from the blood than those
stabilised by poloxamine 908 (FIG. 5). The rapid fall in blood level seen
for both curves can be attributed to the presence of the small quantities
of free iodine that was administered. A kinetic analysis of the data
(first order) indicates that the egg lecithin stabilized emulsion is
cleared from the blood with a half life of about 5 mins while the P-908
stabilized emulsion is cleared with a half life of about 208 minutes. The
plateau level of activity seen for the egg lecithin data reflects the fact
that the emulsion is being metabolised and iodinated breakdown products
are being released into the plasma to give a more or less steady state
level.
The activity recorded in the liver of an animal after the administration of
a colloidal system will include activity resulting from the uptake of
those particles by liver cells (most probably the Kupffer cells) as well
as normal circulating activity as part of the blood pool. This
approximates to 25%. Thus in the studies conducted with poloxamine 908 it
can be concluded that all the activity recorded in the liver (spleen)
region is due to circulating unsequestered emulsion and that the block
copolymer effectively prevents liver uptake of the emulsion.
The results of the study confirm the investigations conducted by using
polystyrene microspheres coated with the block copolymer poloxamine 908
that such systems are largely ignored by the liver and are kept in
circulation for an extended period of time. Such systems could have great
advantages for the delivery of pharmacological agents, where uptake of
emulsion particles by the liver needs to be avoided to prevent adverse
reactions and side effects.
EXAMPLE 3
Targeting of Colloidal Particles to the Bone Marrow using the Block
Copolymer--Poloxamer 407
The purpose of this study was to evaluate the extent and site of diversion
of the poloxamer 407 coated polystyrene particles in the intact animal
model. This material has the ability to deliver model colloidal particles
selectively to the bone marrow.
Methods
Polystyrene particles (60 nm in diameter) were purchased from Polyscience
(Northhampton, UK). The particle size was confirmed using photon
correlation spectroscopy (PCS). The particles were surface labelled with
iodine-131 as described previously. Poloxamer 407 (average MW 10500) was
provided by Ugine Kuhlman Ltd., Bolton, UK, and used as received.
The labelled polystyrene particles were incubated for 24 hours with a 2%
w/v solution of poloxamer 407 providing a surface coating layer of 123
.ANG. thickness as measured by PCS.
Groups of New Zealand White rabbits (3 kg) (n=3) were injected
intravenously via the marginal ear vein with either uncoated polystyrene
particles (0.3 ml, 4.times.10.sup.13 particles, 3 MBq activity of
particles coated with poloxamer 407 (0.6 ml, 4.times.10.sup.13 particles,
3 MBq activity). Particles coated with poloxamer 407 were administered as
the incubation mixture, uncoated particles in distilled water.
Blood samples were taken at suitable intervals and the activity measure
using a gamma counter. The distribution of the labelled particles in the
body was followed by gamma scintigraphy. Dynamic and static images of the
liver, spleen region and the left hind leg were analysed by creating
regions of interests and compared to the whole body activity. Eight days
after administration the rabbits were sacrificed and organs removed. Total
activity in selected organs, blood, femur and remaining carcass was
determined using a large sample volume gamma counter.
Results
Gamma camera scintiscans of the rabbits clearly demonstrated that uncoated
polystyrene particles were largely taken up by the liver and spleen after
injection while the poloxamer 407 coated particles were deposited in the
bone marrow thereby providing a distinct picture of the rabbit skeleton.
Furthermore, no images of the liver/spleen region or other organ regions
could be visualized. (FIG. 6).
The uptake of the uncoated particles by the liver/spleen region occurred
both rapidly and efficiently with 90% of the particles being deposited in
these organs within 2 min. This is illustrated in the liver/spleen
activity-time profiles for the first 15 min after injection (FIG. 7). The
poloxamer 407 coated particles showed a markedly decreased liver/spleen
activity that reached a maximum of 25% after 2 min and then gradually
decreased to a level of 17%. About 10% of this activity can be attributed
to the activity in the circulation (blood pool) and does not represent
particle removal. During the same time period the poloxamer 407 coated
particles were rapidly accumulated in the bone marrow with a half life of
uptake of about 2 min as seen in the activity-time profile obtained by
creating a region of interest around the left hind leg (FIG. 8). In
comparison only background levels of activity were recorded for the same
region of interest in rabbits receiving the uncoated particles. Measured
blood level activities showed that both the uncoated and coated particles
were rapidly removed from the blood-stream. The estimated half lives of
blood clearance correspond quite well with the measured half lives of
uptake in the liver/spleen and the bone marrow, respectively (FIG. 9).
Organ levels measured eight days after administration of the particles show
conclusively that coating the particles with poloxamer 407 leads to a
reduction in lung, spleen and liver uptake. But more importantly a
dramatic increase in the bone uptake is indicated by measured activity in
the femur and the remaining carcass (FIG. 10).
DRUG DELIVERY APPLICATIONS
The particles coated with poloxamine 908 that are retained in the blood
stream could be used to target to other sites in the micro-vasculature,
for example to subsets in the bone marrow, the liver itself, heart,
kidney, lungs and even to tumour cells if the tumour had a vasculature
that allowed extravasation. This type of targeting is termed active
targeting and requires the attachment of a suitable ligand to the particle
or to its polymer coat. Suitable ligands include monoclonal antibodies or
their fragments, apolipoproteins, sugars and lectins.
Drugs that could be administered using particles coated with poloxamine 908
include anti-infectives (for example amphotericin), macrophage activating
agents, antithrombotics, cardiovascular agents (for example
prostaglandins) and anti-leukemia drugs.
The particle coated with poloxamer 407 could be used to direct drugs and
radiodiagnostic agents to the bone marrow. These include
immunosuppressants (cyclosporin), peptide drugs such as colony stimulating
factors and radio-isotopes for diagnostic purposes (e.g. iodine isotopes,
technetium -99 m.
While the example given refers mainly to a model non-degradable particle,
polystyrene, the same concept should work equally well with particles that
will biodegrade in the body. Examples include albumin, gelatin,
polyalkylcyanoacrylates, polylactides, polyglycolides,
polyhydroxybutyrates and their mixtures in the form of copolymers. It also
includes emulsions and phospholipid vesicles.
The coating agent does not necessarily have to be a block copolymer
comprising polyoxyethylene-polyoxypropylene groups as shown in the
example. Other materials that would provide the same type of effect could
be used. Examples include poloxamers, polymaleic acid, polymers that are
esterified to produce suitable hydrophilic and hydrophobic domains as well
as natural materials such as polysaccharides and hyaluronic acid. Polymer
coatings that provide not only a steric barrier but also an electrostatic
barrier are also effective in diverting particles away from the
reticuloendothelial system and materials such as xanthan gum which
consists not only of a hydrophilic chain but also charged carboxyl groups
are a suitable starting point provided it could be attached well to the
surface of the colloidal particle in question. Colloidal particles in the
form of liposomes and emulsions could also be coated with similar types of
material. The results also indicate that the polymeric material tetronic
908 and macromolecules with similar hydrophilic/hydrophobic domains could
also be used as soluble macromolecular carriers for drug molecules by
direct linkage or through degradable spacers and linkages.
Attachment of suitable hydrophilic groups to particles have been achieved
by surface grafting techniques either during the polymerisation process
whereby the particle is produced initially, or by subsequent grafting
methods involving energetic sources such as ultraviolet light and gamma
irradiation.
Poloxamine 908 and Poloxamer 407 (CFTA names) are also available
commercially under brand names TETRONIC and PLURONIC (Registered Trade
Marks) from the BASF WYANDOTTE Corporation 100 Cherry Hill Road, P.O. Box
181 Parsippany N.J. 07054.
TABLE 1
______________________________________
Surface characteristics and phagocytic uptake of
polystyrene particles coated with non-ionic surfactants
Molecular block
Thickness Relative
Average values
of coating
phagocytic
(in moles) layer uptake
Coating agent
EO PO EO A %
______________________________________
None -- -- -- 0 100.0
Poloxamer 108
46 16 46 58 100.4
Poloxamer 184
13 30 13 24 129.3
Poloxamer 188
75 30 75 76 95.4
Poloxamer 217
52 35 52 58 87.6
Poloxamer 235
27 39 27 35 86.5
Poloxamer 237
97 39 97 132 47.0
Poloxamer 288
122 47 122 130 56.5
Poloxamer 335
38 54 38 53 66.7
Poloxamer 338
128 54 128 158 36.7
Poloxamer 407
98 67 98 154 21.6
Poloxaming 908
-- -- -- 134 69.5
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TABLE 2
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Blood Level Activity 15 mins and 1 hour after
Administration of Uncoated and Coated Polystyrene
Microspheres to Rabbits
percentate of initial
dose in blood (.+-.SEM)
15 min 1 hour
______________________________________
Polystyrene 4.0 (.+-.0.4) 3.0 (.+-.0.1)
microspheres (PM)
PM coated with 65.5 (.+-.4.1) 60.0 (.+-.4.1)
poloxamine 908
______________________________________
TABLE 3
__________________________________________________________________________
Deposition of Uncoated and Coated Polystyrene Microspheres in the
Various
Organs 8 days after Intravenous Administration in Rabbits. The Values
are
Expressed as Percentage of total Activity (.+-.SEM)
Lung Heart Kidney
Spleen
Liver Carcass
__________________________________________________________________________
Polystyrene
0.15 .+-. 0.01
0.11 .+-. 0.01
0.22 .+-. 0.02
1.45 .+-. 0.20
59.5 .+-. 6.9
38.6 .+-. 7.1
microspheres
(PM)
PM coated with
0.51 .+-. 0.03
0.22 .+-. 0.01
0.34 .+-. 0.03
0.93 .+-. 0.19
30.2 .+-. 5.5
67.9 .+-. 5.7
Poloxamer 338
PM coated with
2.50 .+-. 0.80
0.20 .+-. 0.01
1.50 .+-. 0.20
1.20 .+-. 0.10
18.9 .+-. 3.2
73.7.+-. 2.4
Poloxamine 908
__________________________________________________________________________
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