Download Predinical Experiences with Magnetic Drug Targeting: Tolerance

ICANCER RESEARCH56. 4694-4701. October IS, 1996]
Predinical
Experiences
with Magnetic
Drug Targeting:
Tolerance
and Efficacy
Andreas Stephan Lübbe,'Christian Bergemann, Winfried HUhnt, Thomas Fricke, Hanno Riess,
Jeffery
Walter
Brock,
and Dieter
Huhn
Department of Medicine (Hematology and Oncology). Virchow Medical School. Humboldt-Universität zu Berlin, Augustenburger Platz I. 13353 Berlin (A. S. L, C. B., W. H.,
7,.F.. H. R., D. H.J and Department ofBiological Sciences. Murray State University, Murray, Kentucky42071 (J. W. B.)
ABSTRACT
Although
slte-spedflc
direction
of drugs
within
an organism
would
benefit patients with many diseases, active drug targeting Is dinically not
yet possible. To overcome some of the problems associated with active
drug targeting, we have developed a magnetic fluid to which drugs,
cytokines, and other molecules can be chemically bound to enable those
agents to be directed within an organism by high-energy magnetic fields.
In the first part of this study, various concentrations ofthe magnetic fluid
were tested in rats and Immunosuppressed nude mice with regard to
subjective and objective tolerance. In the second part, the same parame
ters were evaluated after administration
of the ferrofluid to which epiru
bicin (4'-epldoxorublcin)
was chemically bound. Finally, two forms of
therapy with the magnetic fluid were tested: tumor treatment by median
ical occlusion
with the ferrofluid
in high concentradonsi
and magnetic
drug targeting, using small amounts of the ferrofluid as a vehicle to
concentrate epirubicin locally in tumors. As a result, the ferrofluld did not
cause major laboratory abnormalities
there was no LD,@. With very high
led to complete
tumor
responses
in an experl
mental human kidney as well as in a xenotransplanted colon carcinoma
modeL Thus, the magnetic
fluid is a safe agent, which can be used in
different ways for local forms of cancer treatment In conjunction with
high-energy
magnetic
magnetic carriers in vivo, and the need of strong-enough
magnets with
constant field gradients, to name but a few, there was concern regard
ing the long-term deposition of magnetic particles in the organism, in
addition to discouraging data from large animal experiments and
general
concentrations of the ferrofluid, animals showed lethargy for 1—2
days.
There were no Intolerances with the epirubicin-bound ferrofiuld as well.
Both forms of treatment
of the particle size and the surface characteristics. Many attempts have
been made to formulate various forms of alternative drug delivery
methods, yet they are all in preclinical stages.
Although magnetically controlled, targeted chemotherapy had been
experimentally tried with various systems (magnetic emulsions, mag
netic starch microspheres, magnetic erythrocytes, and magnetic albu
mm microspheres), never has a patient been treated with such a
system (8—10).There have been too many problems that needed to be
overcome; apart from a lack of data suggesting easy large-scale
production of the magnetic drug carrier, a lack of data supporting
reproducibility in the making of the system, aggregation of some
fields.
obstacles
with regard
to regulatory
approval
tal steps, we have produced the first ferrofluid to which drugs can be
bound directly. Its advantage is the independence
and the excellent
INTRODUCTION
There is a long-existing
systemic
problems
wish to treat local problems locally and
with systemic
medical
therapy.
However,
amount of drugs needed (biological
and economic
impact) and the
opportunity
to use drugs that otherwise would not be able to be given
to the patient (pharmacological impact; Ref. 3).
Although many attempts have been made to find a clinically ap
plicable way to perform drug targeting, so-called active drug targeting
is currently not available for any medical indication. It requires
guidance of the particular drug to the target cells in a manner that
differs from its normal distribution characteristics. Conjugation of
drugs to antibodies specific to target cell antigens is one example that
is clinically being explored (4, 5). Passive drug targeting, on the other
hand, is used clinically in the form of liposomes and other vehicles
that incorporate the drug component and release it according to
passive rules of distribution (6, 7). Those rules are mainly a function
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whom
requests
for
reprints
should
under
of a third vehicle
the influence
of magnetic
the organism,
it should
be possible
to concentrate
it in tissues
or tumors that are located at the body surface by externally applied,
high-energy
magnetic
fields.
By these
means,
two modes
of action
seem possible; therefore, we investigated: (a) if the amount of the
ferrofluid were high enough, one could expect mechanical occlusions
of tumor blood vessels and some therapeutic benefit; and (b) if the
minimal amount of the ferrofluid was used as a vehicle to direct a
cytostatic
drug to a tumor,
then drug concentrations
could
result
in
tumor responses that would otherwise not cause those responses with
systemic application.
Therefore, it was the purpose of the following studies to test
whether the magnetic fluid in physiological as well as supraphysi
ological concentrations was tolerated well in two animal models,
Sprague-Dawley rats and immunosuppressed mice. We examined
behavior, laboratory values, tissue specimens, and survival. Also, we
wanted to know whether the ferrofluid could be used for magnetically
controlled (i.e., magnetic field-dependent) localization within the or
ganism and, more specifically, whether magnetically bound epirubi
cm, a well-known and widely used anticancer drug, could be directed
to tumors that were localized at the body surface.
MATERIALS
AND METHODS
The Ferrofluid
Received2/21/96;accepted8/15/96.
6-8, 33175 Bad Lippspringe, Germany.
in vivo stability
fields.
If the ferrofluid were physiologically well tolerated and directable
within
if the
local disease problem is within an organism and inaccessible to
conventional local treatment forms (surgery, radiation therapy, and
topical application of antibiotics) systemic treatment is often used to
treat local problems. This, in turn, requires administration of large
amounts of drugs, most of which are metabolized by normal tissues
(1 , 2). For drugs with a low therapeutic index, this causes a series of
problems. Site-directed drug targeting could circumvent this problem.
Other advantages of drug targeting are the potential to reduce the
I To
and the econom
ics of the therapy (8). Taken together, numerous experiments on small
animals have not lead to further exploration of the idea to direct drugs
within large organisms by means of magnetic fluids in conjunction
with magnetic fields.
Some of the above-named problems were directly associated with
the magnetic fluid or ferrofluid itself. Through a series of experimen
be addressed,
at Cecilien-Klinik,
Cecilienallee
The ferrofluid was obtained from Nano-Technologies GBR (Berlin, Ocr
many). It was a colloidal dispersion, made by wet chemical methods from iron
oxides and hydroxides into special multidomain particles. Those particles were
specially (not randomly) arranged to possess advantages with respect to
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PRECLINICAL EXPERIENCES WITH MAGNETIC DRUG TARGETING
directability under the influence of magnetic fields in vivo. The particles were
surrounded with anhydroglucose polymers to stabilize the magnetic particles
under various physiological conditions. In addition, the surrounding polymer
Tumors
was used for chemoadsorptive binding. Because the binding was reversible,
desorption of the drug that had been bound to the surface occurred according
to the physiological environment (pH, osmolality, and temperature). Thus, the
desorption of drugs took place by competitive forces through blood electro
lytes and could
be varied
according
to the specific
need.
In the experiments
described below, desorption ofepirubicin took place 30 mm after the measured
intravasal availability of the magnetic particles. Thus, it was assured that the
drug could act freely once it had been placed in the tumor by the magnetic
field. The ferrofluid was isotonic, with a viscosity close to that of water. The
dispersion contained 1.5% particles, i.e., 15 mg (0.24 mM)iron oxide in 1 ml.
The particle diameter was 50—150am. The pH was 7.4, the color was black,
and the odor was neutral. The magnetic particles occupied 1.5% of the total
weight; the rest of the ferrofluid was water. The particles consisted of Fe304
and contained 60% pure iron, such that there was a total iron content of roughly
6 mg/ml ferrofluid (German potent 19624426.9).
The Magnetic Field
For the animal experiments, high-energy permanent magnets were used that
were made of rare earths (neodymium being the primary agent). They were
column or block shaped and consisted of discs or blocks with variable thick
nesses. Thus, the magnets could be arranged most tightly around the individual
configuration of the tumors. The magnets never compressed the tumors and
created a magnetic strength of up to 0.5 tesla but at least 0.2 tesla (depending
on the size of the tumor).
Epirubicin
Epirubicin (4'-epidoxorubicin;
Farmorubicin) was a gift from Pharmacia. In
the respective experiments, the dry substance was diluted with the ferrofluid
instead of isotonic saline solution according to standard procedures within 15
mm prior to administration.
and Technique
of Implantation
The tumors that were used in the experiments were either a malignant and
aggressive metastasizing adenocarcinoma of the colon or a hypernephroma.
Both tumorshadbeen removedfrompatientsandcultivatedwithinnudemice
for up to 20 generations. Both tumors were rather undifferentiated, according
to 03. As described in detail in other articles (11, 12), small tumor pieces
(volume, 5—15
p3) of a donor animal were prepared and put into Ringer lactate
solution. After injection of 10 @.tl
isotonic saline under the dermis of the dorsal
ear surface, a small s.c. pocket (3 mm long and 1 mm wide) was created. One
of the tumor pieces was cut to a size of approximately 1 p3, a volume
equivalent to a sphere with a diameter of 1.25 mm, and was introduced into the
ear pocket before the 1-mm incision was sewn with a 10-0 silk suture. In other
experiments, larger tumor pieces (50 @sl)
were transplanted s.c. bilaterally into
the abdominal region.
Experimental PrOtOCOlS
Rat Experiment
1: Weight
and Behavior.
Of 23 rats, four subgroups
were formed. Subgroup 1 (n 4) received isotonic saline solution (5% of the
estimated blood volume, which was estimated to be 10% of the body weight).
Subgroup 2 (n = 7) received the ferrofluid (5% of estimated blood volume).
Subgroup 3 (n = 6) received the ferrofluid (5% of estimated blood volume)
bound to epirubicin (1 mg/kg body weight). Subgroup 4 (n
6) received an
injection of 1 mg/kg body weight epirubicin only (volume also 5% of the
estimated blood volume). Animal behavior and weight were documented daily
for up to 4 weeks.
Rat Experiment 2: Laboratory Values. In six subgroups (36 rats), em
phasis was put on hematological and chemical laboratory values. Subgroup 1
(n
6) received the ferrofluid in a low dose (0.5% of estimated blood
volume). Subgroup 2 (n = 6) received the ferrofluid in a high dose (5% of
blood volume). Subgroup 3 (n 6) received 1 mg/kg body weight epirubicin;
subgroup 4 (n 6) received 10 mg/kg body weight epirubicin; subgroup 5
(n 6) received 1 mg/kg body weight epirubicin bound to the ferrofluid; and
subgroup 6 (n = 6) received 10 mg/kg body weight epirubicin bound to the
ferrofluid. The volume of injection in the last four subgroups was 0.5% of the
estimated blood volume in each experiment. Animal behavior, weight (not
shown), and laboratory values were obtained prior to and I, 2, 7, 14, and 35
Animals
Overall, 71 male Sprague-Dawley
rats were used for the studies. They were
obtained from Bomholtgard (Ry, Denmark). Animals were maintained on a
controlled diet of Purina Rat Chow and were given water ad libitwn in an
American Association for the Accreditation of Laboratory Animal Care
approved animal care center. They were housed in an environmentally con
trolled room with a 12-h light-dark cycle until body weights ranged between
180 and 280 g.
and were acclimated
under laminar-flow
conditions
an overdose of ketamine-HC1, and relevant organs were histologically
at room
temperatures between 24-26°Cand a humidity of6O% in the same animal care
center. The mice were allowed a standard laboratory diet (3-Altromin 1320;
exam
med with standard procedures for H&E or potassium hexacyanoferrat (staining
for iron).
Mouse Experiment
Overall, 166 male athymic nude mice (NMRI-nu/nu), weighing between 20
and 28 g at the age of 6—12weeks, were used for the studies. These animals
were purchased from the same company as above 1—2
weeks prior to the
experiments
days after therapy.
Rat Experiment 3: Histology. In 12 rats, the ferrofluid (2% of the esti
mated blood volume) was injected i.v. over 1 mm into the tail vein. Four, 12,
18, and 65 days later, three of the animals were sacrificed in each subgroup by
1: Survival.
In 13 subgroups, animals were subjected
to an injection of either isotonic saline (5% of estimated blood volume; n 4)
or the ferrofluid [ranging from 0.1% of the blood volume (n = 4) to 1%
(n 5), 5% (n 6), 10% (n = 5), and 20% (n 4) of the blood volumej.
Subgroup 7 (n = 5) received 0.5 mg/kg body weight epirubicin; subgroup 8
(n
4) received
0.9%
saline
solution;
and subgroup
9 (n
6) received
mg/kg body weight epirubicin bound to the ferrofluid. To subgroup 10 (n
1
6),
were anesthetized with sodium pentobarbital (45 mg/kg i.p.). For all other
0.5 mg/kg epirubicin bound to the magnetic fluid was given; subgroup 11
(n
5) received 1 mg/kg body weight epirubicin; subgroup 12 (n
4)
received 10 mg epirubicin/kg body weight bound to the ferrofluid; and sub
group 13 (n = 4) received 10 mg epirubicinlkg body weight only. The volume
of each injectant in the last 7 subgroups was 0.5% of the estimated blood
volume. Overall, 62 mice were used for those survival studies. The observation
experiments, animals were lightly anesthetized with ketamine-HCI (45 mg/kg
body weight) and xylacin (6 mg/kg body weight). Rectal temperature was
period was terminated after 12 weeks.
Mouse Experiment
2: Tumor Embolization.
continuously monitored and maintained between 36 and 37°Cby a heating
lamp, which was positioned above the animal. Respiration rates were also
monitored.
The various compounds (epirubicin, the ferrofluid, and the magnetic epiru
bicin) were injected into the tail veins of the animals, which sometimes had
subgroups (subgroup I, colon carcinoma, n 6; subgroup 2, hypernephroma,
n = 6, implanted into the outer ear) received the ferrofluid (10% of the
been
Mouse Experiment 3: Tumor Treatment with Epirubicin.
In four sub
groups with a total of 16 animals, two concentrations of epirubicin (I and 10
mg/kg body weight) were administered i.v. into the tail veins of the animals,
Altromin, Lage, Germany) and acidified water ad libitum. For the mouse as
well as rat experiments, food but not water was withdrawn 12 h before each
experiment. All animals were handled according to the NIH 1978 Guide for the
Care and Use of Laboratory Animals. For tumor implantation, mice and rats
lightly
anesthetized.
Histological
specimens
were
sometimes
Each mouse of the two
estimated blood volume). A magnetic field was applied over the tumor for 20
obtained
after injection of an overdose of the anesthetic. Specifically, organs were
quickly removed after the animals had been sacrificed, embedded in formalin,
and processed according to standard procedures for staining with H&E or
potassium hexacyanoferrat for iron, usually within the next 48 h.
mm, including the time of injection, roughly 1 mm. Then, the tumor was
measured manually in three axes over the next 14 days.
which were carrying large tumors (colon and kidney cancers) under the
abdominal skin.
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PRECLINICAL EXPERIENCES WITH MAGNETIC DRUG TARGETING
.n
Sal
Fig.
1.
Weight
of
Sprague-Dawley
rats
(378 ±12 g baseline weight) after application of
isotonic saline solution (0), magnetic fluid (MF;
5% of the estimated blood volume; •),epirubicin
>1@@
all
(Epi: 1 mg/kg body weight; 0), and magnetic epi
rubicin (5% of estimated blood volume; 1 mg/kg
body weight, @).Data presented as means; bars,
a
.@
@-
U
SEM. No differences among the groups were found.
0
2
4
6
8
10
12
14
16
18
flue (days aftertrealment)
Mouse
Experiment
4: Magnetic
Drug
Targeting.
The volume
rats, as indicated for each group, increased or remained at baseline
of xeno
transplanted colon and kidney cancers was measured daily over 10 days in six
levels according to normal growth rates over the period of 18 days, no
subgroups after treatment with I mg epirubicin/kg body weight bound to the
ferrofluid and directed to the tumor with a magnetic field (colon, n = 6; kidney
cancer, n = 5) or not directed to the tumor with a magnetic field (colon,
n = 10; kidney cancer, n = 6) or with no treatment of the two tumor entities
at all.
Mouse Experiment
5: Control
Studies.
7; kidney cancer, n = 6) while a magnetic field was
applied for 20 mm, in contrast to two other subgroups, in which the procedure
was identical, except that a magnetic field was not applied (n 6 in both tumor
groups).
Statistics
whether
Laboratory
In four subgroups, the ferrofluid
(0.5% of the estimated blood volume) was administered in the two tumor
entities (colon, n
matter
RESULTS
The first part of the study put emphasis on the tolerance of the
ferrofluid or the magnetically bound epirubicin. Body weights of the
saline
solution
(sham-treated
values for the animals
of experiment
2 demonstrate
Day 0 (0.5/5%)―Day
1 (0.5/5%)Day
group
(10 mg/kg
body
weight
epirubicin),
but in the sur
2 (0.5/5%)Day
7 (0.5/5%)Day
14 (0.5/5%)Day
35
133/143141/146143/142141/145141/145146/141Glucose
(M)
247/248252/194261/180276/229264/189169/195Creatinine
(mg/dl)
0.4/0.450.4/0.450.4/0.40.3/0.450.35/0.40.3/0.4Albumin
(mg/dI)
2.3/2.92.2/2.92.5/2.92.7/2.73.0/2.92.7/2.7Iron
(g/dl)
30.7130.238.7/25.829.7/33.734.0/30.839.0/39.032.0/38.0Bilirubin
(psi)
0.3/0.150.6/0.20.1/0.20.1/0.20.1/0.20.15/0.9Cholesterol(mg/dl)
(mg/dl)
96/11698/106117/126110/110112/116100/110ALT
(units/liter)
44/39
GOT6
2/334/38
7/430/44
3/4AP'(units/liter)
263/239210/249184/233195/213216/247180/222Lipase
(units/liter)
<50/<50<50/<50<50/<50<50/<50<50/<50<50/<50Hemoglobin(g/dl)
(units/liter)
4/442/44
2/349/45
2/445/38
14.0/14.912.6/13.411.0/12511.6/14.313.3/12.315.1/15.0Leukocytes
(0/liter)
17.0/14.01
1.0/17.716.0/17.015.1/16.512.1/10.68.3/14.6l'hrombocytes
951/1006706/1048834/6421258/12221321/727848/936a
(G/liter)
Ferrofluid
as percentages
of estimated
blood volume.
b
Gamma-glutamyltransferase.C
Alkaline
that
viving animals there were also significant drops in the number of
WBC, platelets, and hemoglobin (Table 2). This basic pattern did not
change significantly with regard to the absolute values in the groups
in which epirubicin at both concentrations was bound to the ferrofluid
(Table 3). All other organ functions we measured seemed to be intact.
The histological data revealed that the magnetic particles accumu
lated in the liver and spleen, as expected, without causing significant
hepatosplenomegaly (by wet weight analysis), whereas they were not
Table 1 Meanlaboratory values over time prior to and after application of the
ferrofluidLaboratory
value
(0.5/5%)Sodium
received
relevant hematological and blood chemical values did not change
from baseline after injection of different amounts of the ferrofluid (0.5
and 5% of the estimated blood volume; Table 1). Epirubicin, however,
did cause changes in hematological parameters, as expected. Although
this was not particularly striking in the low-dose range (1 mg/kg body
weight epirubicin), not only was there a significant mortality in the
high-dose
In general, data were reported as means ±SEM. Means ±SEM of the
tumor volumes were calculated as baseline values and for changes over time.
Baseline data were analyzed by one-way ANOVA to test for any changes at the
P < 0.05 level and the Bonferroniprocedure for multiple comparisons.
the animals
rats), the ferrofluid in high doses, or epirubicin with and without being
attached to the magnetic fluid (experiment 1; Fig. 1). Overall, there
were no statistical differences among the groups.
phosphate.
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PRECLINICAL
epirubicinLaboratory
mg)Sodium
EXPERIENCES
WITH MAGNETIC
DRUG TARGETING
Table 2 Mean laboratory values over time prior to and after application of
Day I (1/10 mg)
Day 2 (1/10 mg)
Day 7 (1/10 mg)
Day 14 (1/10 mg)
139/143Glucose
(mM)
145/139
140/138
141/140
138/140
140/138
187/—Creatinine
(mg/dl)
221/177
186/166
176/227
171/185
167/155
0.5/0.5Bilirubin(mg/dl)
(mg/dl)
0.4/0.4
0.5/0.4
0.5/0.5
0.4/0.4
0.4/0.5
0.1/0.1ALT
0.8/0
0.1/0
0.1/0
0.1/0.1
0.1/0.1
(units/liter)
34/33
34/18
46/26
30/29
39/22
256/—Lipase
APb
(units/liter)
<50/<50Hemoglobin
(units/liter)
14.9/13.7Leukocytes
(g/dI)
8.0/8.5l'hrombocytes
(G/liter)
866/1600a
(G/liter)
184/254
<501<50
14.9/14.8
9697.5
731/844
250/206
<50/<50
14.1/13.9
8.8/8.6
666/818
247/—
<501<50
13.2/13.1
9.2/5.7
744/678
215/196
<50/<50
13.2/10.9
9.0/2.9
972/529
229/206
<50/<50
13.6/8.7
7.4/4.3
792/428
Epimbicin
value
doses
Day 0 (1/10 mg)―
in mg/kg
Day 35 (1/10
35/23
body weight
phosphatase.found
b Alkaline
themeasured;
in other organs. We noted a significant (semiquantitatively
tumor)those data
not shown)
decrease
in the number
of particles
over 7 days compared with treatment with no magnetic field (i.e.,
in
magnetic
betweenfar
two organs over the 65 days. Organ structure was not altered, as
types.No
as histological criteria were concerned (experiment 3; Table 4),
causedmice,
matter which concentration of the ferrofluid we injected into the
bloodobservation,
there was no increased mortality rate. After the sixth week of
drug was given, but no magnetic
field attached
to the
or no treatment whatsoever (Fig. 6). There was no difference
the two tested tumor
Fig. 7 shows that the same amount of the magnetic fluid that
successful magnetic drug targeting (0.5% of the estimated
epirubicincardiovascular
one animal in each high-dose group died, probably of
volume) did not cause significant tumor responses when
nonresponse,those
collapse due to sepsis (mouse experiment 1; Fig. 2). In
role.animals
groups in which the high dose of epirubicin was administered,
had not been attached to the ferrofluid. Thus, for this
application of a magnetic field did not play any
somewhatlater,
died rapidly;
in the low-dose
groups,
they died
around 4—6weeks (Fig. 3). This fact was independent of
epirubicin
includingthebeing bound to the ferrofluid. In all other groups,
by(0.5
ones in which a low concentration of epirubicin was administered
mg/kg body weight), animals survived over the observation
period
weThe
(Fig. 3).
magneticlization
second part of this research focused on the mechanical embowell?by by the ferrofluid after injection and concentration in the tumor
showtype,
means of an external magnetic field. Independent of the tumor
there was a rapid and consistent decrease of the tumor volume
within
specialIt
14 days after treatment (Fig. 4).
thatinwas impossible to reproduce this tumor response in the animals
specialthe
which only epirubicin was given. Although tumors did respond to
irona
high dose of epirubicin (10 mg/kg body weight), this was only for
whichshortly
brief period (several days), and most animals of this group died
DISCUSSION
similarbasically
thereafter.
the effect
According
to the
baseline
volumes,
there
The present study demonstrates good tolerance of the ferrofluid
the animals and effective tumor therapies with both mechanical ob
struction by high concentrations of magnetic particles and what
call magnetic drug targeting, using low concentrations of the
fluid. Is it surprising that the ferrofluid was tolerated
Both the laboratory values and the histological observations
that organ function was not significantly altered acutely and chroni
cally. The magnetic fluid that was used in this study was a
colloidal dispersion, which consisted of multidomain particles
were manufactured such that the particles were aligned in a
manner on an external magnetic force. The particles consisted of
oxide and hydroxide and, therefore, were made of material for
was
forthe
an unaltered growth characteristic of the tumors over time in
Althoughall
groups that received the low dose of epirubicin or no treatment at
wasMagnetic
(Fig. 5).
drug targeting demonstrated
studies,Table
significant tumor responses
on live
organisms
is well
known.
Specifically,
ferrofluids have been developed as ferrimagnetic contrast agents
diagnostic nuclear magnetic resonance purposes (13, 14).
the latter compounds are considerably different from the fluid that
used in this study, they also contain iron cores. In preclinical
3 Mean laboratory values over time prior to and after application of magnetic epirubicin
DayO
Day]
Day2
Day7
Dayl4
Day35
(1/10 mg) + 0.5% MFa (1/10 mg) + 0.5% MF (1/10 mg) + 0.5% MF (1/10 mg) + 0.5% MF (1/10 mg) + 0.5% MF (1/10 mg) + 0.5% MF
Laboratory value
Sodium (mM)
142/140
141/144
145/142
145/143
140/142
142/139
Glucose(mg/dl)
Creatinine (mg/dl)
170/0
0.4/0.4
195/0
0.4/0.4
168/0
0.4/0.4
0/0
0.3/0.3
212/0
0.3/0.3
0/0
0.3/0.4
Iron (@sM)
ALT (units/liter)
28/29
40/43
33/41
36/37
35/57
33/26
37/36
40/43
24/28
38/43
34/29
41/42
GGTb (units/liter)
2/3
Hemoglobin (g/dI)
Leukocytes
(G/liter)
Thrombocytes (G/liter)
a Epirubicin
doses
in mg/kg
2/2
13.4/14.8
10.5/1
1.0
981/809
body weight.
13.7/12.8
11.1/16.3
909/628
MFF, magnetic
ferrofluid
3/3
4/3
13.3/12.5
9.7/8.7
1022/749
12.8/12.9
8.5/1
1.5
775/696
given at 0.5% of the estimated
blood
4/4
13.5/0
10.
1/0
123/0
3/1
15.1/14.2
13.6/10.7
515/694
volume.
b Gamma-glutamyltransferase.
dataLiverLungKidneySpleenHeartAnalysis
Table
4 Histological iron deposition in some organs at varioustimes after injection ofthe ftrrofluid
after 4 days
Analysis after 12 days
Analysis after 18 days
Analysis after 65 days
—a
@
÷
@,much;
+ +, moderate;
(2% of the estimated blood volume): semiquantitative
++
+ + +(‘
+++
++
++—
+++
—
——
—
—+
+++
—
—
+ +—
and —,little iron deposition.
4697
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PRECUNICAL
EXPERIENCES
WITH MAGNETIC
DRUG TARGETING
90
90
70
60
Fig. 2. Long-term survival of nude mice (percentage of respective
group) after increasing volumes (percentage of estimated blood vol
ume) of magnetic fluid (MF) or isotonic saline solution (NaCl). Data
represent the means,.
a90
40
—0—NaCI
30
——MF
0.1%
—D--MF1%
——MF5%
20
—@—MF
10%
—@—MF
Z@%
10
0
0
1
2
3
4
6
6
7
8
9
10
11
Thtie (viaeks after treabnent)
dose escalation experiments with those contrast agents have shown no
harmful effects on organ function, as reflected by laboratory values
and other organ function tests (15, 16). In animal and preclinical
studies, iron concentrations far higher than those used in this study
had been tested. One main difference between ferrofluids used for
diagnostic
purposes
and the one described
in this article was the
particle size. Although the particle size in the ferrofluid used in this
study is 10 to 50 times higher than that of ferrimagnetic contrast
fluids, it was safe. We saw no accumulation in the lungs or any
clinical signs of respiratory problems. Only in supraphysiological
dose
ranges,
in which
10—20% of the blood
volume
was infused
within a relatively short time, subjective responses were obtained in
the animals, characterized by lethargy for 12—24h and resistance of
food uptake. Also, because of the relatively
enormous
iron load,
discoloration
of the animals was noticed for about 1 week, symptoms
quite similar to those noted in preclinical studies by Van Hecke et al.
Fig. 3. Long-term survival (percentage of respective group) of nude mice after
increasing doses of epirubicin (Epi) or epirubicin (all applications in a volume
estimated to be 0.5% of the blood volume) bound to the magnetic fluid (MF).
High and intermediate mortality resulted with 10 and I mg epinibicin/kg body
weight, respectively. Data represent means; bars. SEM.
(16). Taken together, the ferrofluid that was used in this study was
tolerated well from a clinical standpoint, as shown by the laboratory
and histological data. Uptake and successive elimination of iron
particles by the reticuloendothelial system are well-known character
istics of this system, which were confirmed by the histological data.
One crucial characteristic of the ferrofluid was its carbohydrate
coating and thus, on the one hand, its in vitro stability over months
(i.e. , no sedimentation of the magnetic particles) and, on the other, the
capacity for adsorptive binding of many different drugs (German
patent
19624426.9).
An important
feature
of the ferrofluid
was its
ability to separate from the drug whenever necessary. For drug
targeting purposes, it was the intention to use the ferrofluid as a
vehicle to achieve relevant concentrations of the drug within the
tumor tissue. Once localized at the site of choice, desorption must
occur, by which the drug leaves the vehicle and can freely act on the
target tissue. Because anthracyclines treat a wide spectrum of ma
I
0
1
2
3
4
6
6
7
8
Time (v@aeksaft@ trealrnent)
4698
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9
10
PRECLINICAL
EXPERIENCES
WITHMAGNETICDRUGTARGETING
10000
1000
Fig. 4. Tumor regression after mechanical obstruction
by the ferrofluid
(10% of the estimated
blood volume)
in
100
conjunctionwitha magneticfieldlocalizedat the tumor
at the body surface (20 mm). Co colon cancer (•,U);
RC, renal cell carcinoma (0, 0). Data represent means;
bars, SEM; significant difference, P < 0.05 from base
I
line.
10
—0-- RC, therapy
—4—-Go,
therapy
—O--RC,control
—U--Co control
I —
12
14
16
18
20
22
24
26
28
30
line (days after tianor @npIantatIon)
in
7 and 14 days. These data confirm what is known from the literature
some malignant diseases, such as breast carcinoma and soft-tissue sar
(19, 20), and it did not make any difference whether the ferrofluid was
bound to epirubicin or not. For ethical reasons, a magnetic field was
not applied in rats that were not carrying malignant tumors. Thus,
drug-targeting experiments with potentially high toxic concentrations
in healthy tissues of live animals were not performed in this animal
model. It was, therefore, not possible to demonstrate any change in
blood cell counts as a response to drug targeting with epirubicin doses,
which, given systemically, would cause blood cell nadirs.
lignomas,
and because
there is a positive
dose-response
relationship
comas, epirubicin (4'-epidoxorubicin) was used as the anticancer agent in
this study (17, 18). Therefore, epirubicin and magnetically bound epiru
bicin were also tested in rats and mice for clinical andlaboratory function.
Epirubicin
was used in two standard
(19, 20) concentrations
one of 1 mg/kg body weight and a high one of
Although the LD50 for this substance lies
weight, the low dose did not cause major
tolerated well by the animals. The high dose
mortality
rate, but it also led to a typical
(a low
10 mgfkg body weight).
around 3 mg/kg body
abnormalities and was
not only caused a high
hematological
Fig. 5. Tumor response of colon cancer (Co. •,U) and
renal cell carcinoma (RC. 0, 0) after one treatment with 1
and 10 mg epinibicin/kg body weight (Ep:). Data represent
means; bars, SEM.
Through
a series of calculations,
the most practical
route of appli
cation of the ferrofluid was found to be i.v. injection resulting in its
nadir between
I
0
2
4
6
8
10
12
14
16
18
20
Time (days after treatment)
4699
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22
PRECLINICAL EXPERIENCES WITH MAGNETIC DRUG TARGETING
10000
1000@
Fig. 6. Tumor regression with magnetic drug-targeting [magnetic
fluid (MF), 0.5% of the estimated blood volume; I mg epirubicin
100
(Epi)/kg body weight; 20-mm magnetic field application (Dl)]. Colon
cancer (Co. •,U) and renal cell carcinoma (RC. 0, 0) completely
disappear within 7 days. Control studies were conducted with no
magnetic field but the same injected drug versus no therapy at all
I
(control). Data represent means; bars. SEM. There was no difference
between the two tumors (P > 0.05).
10
0
2
4
6
8
10
Time (days after treatment)
localization
at different
sites
in the organism.
Also,
we arrived
at
appropriate concentrations of the compound at which drug targeting
and mechanical obstruction of tumor blood vessels predominantly
occur. In the latter experiments, 10% of the estimated blood volume
had been injected into the animals, and a magnetic field had been
applied to 3-week-old tumors (estimated volume, 10% of the body
volume). In 7 to 14 days, the tumors underwent a series of reproduc
ible changes, leading from initial discoloration to a continuous shrink
ing with a dry and black appearance at the surface to a complete loss
of the tumors. We interpret those stages as a concentration of the
magnetic
particles
in the tumor-feeding
vessels
and the successive
necrosis over the next several days. Therefore, it was possible to cause
complete and lasting tumor remissions by mechanical obstruction in
two different malignant human tumors. In contrast to other forms of
mechanical obstruction (such as the direct injection of particles into
the surgically prepared tumor-feeding artery), the compound was
injected
systemically
and allowed
to distribute
within
the organism.
During this time and the following 15 minutes, a magnetic field that
was applied to the tumor was able to recruit at least that many
magnetic particles that were sufficient to obstruct tumor vessels and
cause necroses. Those tumor obstructions have been confirmed his
tologically and under intravital microscopy (data not shown here).
Roughly 30 years ago, the first attempts were made with vascular
occlusion of tumors and organs by mechanical obstruction with mag
10000-
1000
Fig. 7. Normal growth behavior of colon cancer (Co. closed sym
bols) and renal cell carcinoma (RC. open symbols) to application of the
magnetic fluid (MF: 5% of the estimated blood volume) with and
without a magnetic field (Dl) or compared with untreated controls (A,
@I00
b
L@).Data represent means; bars. SEM.
—O—RC,
MF0.5% + DT
——Co,MF0.5%9-DT
10
—D--RC,MFO.5%
—U--Co,MFO.5%
—6-—
RC, control
—è--Cocontrol
0
2
4
6
8
10
12
line
(days after treatment)
14
16
18 20
22
24
26 28
4700
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PRECLINICAL EXPERIENCES WITH MAGNETIC DRUG TARGETING
netic fluids (21—24). Some of the authors of those studies used
ferrosilicone preparations with limited in vivo stability and low di
rectability with magnetic fields, and others used simple iron particle
suspensions that were placed in mannitol to obliterate cochlear blood
vessels. All of those materials have not fulfilled essential criteria for
long-term in vivo compatibility after systemic injection.
Drug targeting, i.e., the phenomenon of site-directed concentration of
drugs within the organism, has been tried in different diseases and with
many different methods. Especially in cancer therapy, in which chemo
therapeutic
agents with many and severe associated
side effects and low
new form of localized cancer chemotherapy. If it were possible to
obtain high-energy permanent magnets strong enough to direct the
ferrofluid within large organisms, this therapy could be used in
patients with locally advanced
and otherwise
nontreatable
malignomas.
ACKNOWLEDGMENTS
This work was made possible by a gift from W. Miehrendorff
and the
personaleffortof E. Bergemann.
therapeutic indices are available, the protection of normal, healthy tissues
from those drugs and the concentrations
of the drugs exclusively in the
tumor tissues form an important goal. Among those methods are tumor
antigen-directed
and liposomal-encapsulated
drugs (4—7).However,
there are some significant drawbacks with those techniques. Either the
drugs are injected
in the neighborhood
of the target area (sthrch-encap
sulated drugs) and are not available at other body sites, or they are limited
to the liver and spleen, organs of the reticuloendothelial system that
phagocytose drug-containing liposomes. Both approaches are character
ized as so-called passive drug targeting.
Active drug targeting, i.e., the concentration of drugs within the
organism against the local distribution characteristics of the drug, by
magnetically controlled techniques is a roughly 20-year-old attempt to
bind anticancer drugs to magnetically active compounds and to direct
those by means of high-energy magnetic fields to the tumor (8, 25).
Until now, it has not been possible to accomplish that in a satisfactory
way. Either there were technical difficulties in the making of the
magnetic compound, or a third intermediate compound had to be used
to combine the drug with the ferrofluid. This intermediate compound,
however, usually large, denaturated albumin molecules, created many
problems, especially in larger animals; consequently, those experi
ments were stopped in the mid-l980s (8, 9, 25).
The ferrofluid that was used in this study did not necessitate a third
compound as an intermediate. Rather, with this system drugs can be
bound directly to the coating surface ofthe particles. Thus, the drugs can
be directly moved by magnetic forces together with the magnetic parti
des. In addition, the process of desorption of the drug from the magnetic
vehicle within the organism can be precisely determined. This is impor
(ant, because once the magnetic drug is injected into the body and
directed to the tumor by the magnetic field, the drug must dissociatefrom
the magnetic compound to act freely in the tumor. Through a series of
experiments,
we have shown that epirubicin
desorbed
effect with the low concentration
of the
ferrofluid (0.5% ofthe estimated blood volume). Fig. 7 demonstrates that
tumor size did not shrink after injection ofthat amount and application of
the magnetic field for 15 minutes. Also, we showed that epirubicin at the
dose of 1 mg/body weight injected i.v. into rats did not cause tumor
responses.
However, when the two treatments were combined, 1 mg epirubi
cm/kg body weight
was bound to the ferrofluid
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2. Gurney, H., Dodwell; D., Thatcher, N., and Tattersall, M. H. N. Escalating drug
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7. Gabizon, A., Isacson, R., Libson, E., Kaufmann, B., Uziely, B., Catane, R., Ben-Dor,
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and
from the ferrofluid
at 30 mm (half-life). In other words, once the drug was injected into the
organism and a magnetic field was applied to the tumor, 50% ofthe drug
was released under the influence of the magnetic field and was free to act
on the tumor cells. Through anothertest series, we arrivedat a dose of the
ferrofluid of 0.5% of the estimated blood volume. This is considerably
lower than the 10% necessary to mechanically obstruct the blood vessels.
There is no tumor-obstructing
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Preclinical Experiences with Magnetic Drug Targeting:
Tolerance and Efficacy
Andreas Stephan Lübbe, Christian Bergemann, Winfried Huhnt, et al.
Cancer Res 1996;56:4694-4701.
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