Vol. 1, 1369-1374, November 1995 Clinical Cancer Research 1369
Preclinical Toxicity of Liposome-incorporated Annamycin: Selective
Bone Marrow Toxicity with Lack of Cardiotoxicity’
Yiyu Zou,2 Waldemar Priebe, L. Clifton Stephens,
and Roman Perez-Soler
Section of Experimental Therapy, Department of Thoracic/Head and
Neck Medical Oncology [Y. Z., R. P-S.], Department of Clinical
Investigation [W. P.], and Department of Veterinary Medicine
[L. C. S.], The University of Texas M. D. Anderson Cancer Center,
Houston, Texas 77030
ABSTRACT
Annamycin (Ann) is a new bipophilic anthracycline an-
tibiotic with a marked ability to circumvent typical multi-
drug resistance both in vitro and in vivo. Because of its high
affinity for lipid membranes and very low solubility in wa-
ter, Ann has been prepared in a submicron liposome formu-
lation (L-Ann) that is currently being investigated in a Phase
I clinical study. We studied the preclinical toxicity of L-Ann
in mice and beagle dogs and compared it with that of free
Ann in suspension and the parent compound doxorubicin
(Dox). In mice, free Ann was about twice as toxic as Dox
(LD50 after a single i.v. bolus administration, 8.8 versus 19.9
mg/kg; P < 0.01). The liposomal carrier reduced Ann tox-icity by 2-fold (LD50, 15.74 mg/kg for L-Ann versus 8.8
mg/kg for free Ann; P < 0.01). Granubocytopenia was the
main toxicity of Ann, either free or liposome incorporated,
and was much more profound than with an equitoxic dose ofDox as assessed by blood counts and pathological studies. In
chronic mouse studies, L-Ann was remarkably less cardio-
toxic than Dox. Cumulative toxicity with the weekly admin-
istration of a given fraction of the subacute LD10 was mark-
edly higher with Dox than with L-Ann as assessed by body
weight and mortality studies. L-Ann also had less vesicant
toxicity than Dox after intradermal administration in mice.
Beagle dogs tolerated the mouse-equivalent LD10 dose of
L-Ann (1.4 mg/kg) with no side effects, changes in the he-matobogical and biochemical blood parameters, or pathobog-
ical changes. Our results indicate that: (a) L-Ann is more
selectively myebotoxic than Dox and is noncardiotoxic; (b)
the liposome carrier plays a major role in the favorable
toxicity profile of L-Ann; and (c) the standard one-tenth ofthe LD10 should be a safe starting dose for Phase I clinical
trials with L.Ann in humans.
INTRODUCTION
Anthracycline antibiotics are some of the most effective
antitumor agents. However, their use is limited by acute and
chronic side effects and their limited spectrum of activity as a
result of natural or acquired resistance (1). The best known form
of acquired resistance to anthracyclines is typical MDR1,3 the
result of an active, energy-dependent drug efflux mediated by
the membrane glycopnotein P-gbycopnotein, which is ovenex-
pressed in resistant cells (2). Most attempts to overcome MDR 1
have used strategies aimed at blocking P-gbycopnotein function
with resistance modifiers. This strategy has had limited success
because of the toxicities associated with many of the resistance
modifiers and because they block the physiological excretory
function of P-gbycopnotein in many normal organs, thus neduc-
ing the clearance of the drug and increasing its toxicity (3).
Ann is a new bipophibic anthnacycline that was developed
because of its cytotoxicity to MDR1 cells both at in vitro
concentrations and in vivo doses close to those that are cytotoxic
to parental cells (Ref. 4; see Fig. 1 for chemical structure.) The
mechanisms by which Ann is cytotoxic to MDR1 cells are under
intensive investigation. Two hypotheses are being entertained:
(a) that Ann is not a substrate of P-gbycoprotein, and, therefore,
P-glycoprotein does not affect its cellular accumulation; and (b)
that Ann is a potent blocker of P-glycopnotein function at
concentrations below those that cause cytotoxicity, i.e., Ann is
both a resistance modifier and a cytotoxic agent. There is now
evidence that P-glycopnotein can bind to substrates within the
cell membrane (5). Because Ann has a very high affinity for
lipid membranes, this observation might suggest the possibility
of Ann blocking P-gbycopnotein at its transmembrane domains.
However, all information currently available from cellular up-
take and efflux studies of radioactive substrates of P-glycopro-
tein in the presence of Ann suggests that Ann is not a blocker of
P-glycopnotein.
This high affinity for lipid membranes makes biposomes a
natural delivery system for Ann (6). In addition, liposomes
themselves are an attractive delivery system for anthracyclines
because of their demonstrated candioprotective effect (7) and
their potential tumor-targeting properties. Clinical studies with a
stable byophilized pnebiposomab Ann formulation developed in
our laboratory have just started.4 The byophilized powder con-
tains the drug, lipids, and a small amount of surfactant. Lipo-
somes are obtained on the day of use by hydrating the lyophi-
bized cake and mild hand shaking. In this formulation, Ann is
Received 4/13/95; revised 6/26/95; accepted 7/13/95.I Supported in part by NIH Grant CA50270, The Texas Higher Educa-tion Board, and a grant from Argus Pharmaceuticals, Inc.2 To whom requests for reprints should be addressed, at Box 60, TheUniversity of Texas M. D. Anderson Cancer Center, 1515 HolcombeBoulevard, Houston, TX 77030.
3 The abbreviations used are: MDR1, mubtidrug resistance; Ann, anna-mycin; Dox, doxorubicin; L-Ann, liposomal Ann; CPK, creatine phos-
phokinase.
4 Y. Zou, W. Pniebe, and R. Perez-Solen. Lyophilized liposome formu-lation of the non-cross-resistant anthracycline annamycin: effect of
surfactant on liposome formation, stability, and size, submitted for
publication.
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DOXORUBICIN ANNAMYCIN
Fig. 1 Chemical structures of Dox and Ann.
1370 Toxicity of Liposomal Annamycin
incorporated within the biposome membranes. In contrast, in all
liposomab Dox formulations currently in clinical trials, Dox is
encapsulated in the inner aqueous space of the biposomes. We
studied the toxicity of this formulation in mice and dogs and
compared it with that of free Ann in suspension and Dox to
assess the role of the liposome carrier and the structural modi-
fications of the drug in modulating its toxicity. We report here
the results of this study.
MATERIALS AND METHODS
Drug Preparation
Dox (Farmitabia Carlo Enba, Milan, Italy) for injection was
dissolved in normal saline.
Ann was synthesized as previously described (8). The final
purity was >95% as assessed by HPLC. The drug identity was
confirmed by nuclear magnetic resonance. Free Ann was pne-
pared as a suspension in 10% DMSO and 90% normal saline.
L-Ann was obtained by hydrating prebiposomab byophilized
powder containing Ann, dimynistoybphosphatidyb choline, and
dimynistoyl phosphatidyl glycerol (1 mg/35 mg/iS mg). The
manufacture and characterization of the preliposomal powder
are reported elsewhere.4 The hydration step consisted of adding
1 ml of normal saline at 37#{176}C/mgAnn and shaking by hand for
1 mm. The particle size was determined by the light-scattering
technique using a Nicomp sizer model 370 (Nicomp Particle
Sizing Systems, Santa Barbara, CA). The size of the liposomes
was 145 ± 65 nm. L-Ann was physically and chemically stable
for >24 h. All injections were performed <24 h after biposome
formation.
The concentration of the drug used in the different mouse
experiments was dependent on the injected dose; the injected
volume was fixed at 0.1 ml of drug solution/lO g mouse body
weight in all cases. In the dog studies, the concentration of Ann
in the L-Ann preparation was 1 mg/mb.
Mouse Toxicity Studies
Facilities and Personnel. The mouse toxicity studies
were carried out in the small animal facilities of the Department
of Veterinary Medicine at the M. D. Anderson Cancer Center.
The animal facilities are accredited by the American Associa-
tion for the Accreditation of Laboratory Animal Cane. Male
CD1 mice, 7-8 weeks old and 20-22 g each, were purchased
from Harlan Sprague-Dawley (Indianapolis, IN) and housed five
to a cage. The studies were approved by the Institutional Animal
Cane and Use Committee.
Single i.v. Dose Subacute Toxicity (LD10, LD50, and
LD�O). The subacute toxicity of L-Ann was studied in CD1
mice after single i.v. bobus injections and compared with that of
free Ann and Dox. Seven different dose levels were used for
each drug, 10 animals/dose bevel. The maximum dose (resulting
in 100% animal mortality) and minimum dose (resulting in
100% animal survival) were selected in preliminary expeni-
ments. Animals were observed and weighed daily, and animal
deaths were recorded. The experiment was terminated on day
14. The K#{228}rbermethod was used to calculate the lethal doses
(9). The experiment was performed twice.
Single i.v. Dose Myelosuppression. CD1 mice (6-8/
group) were given single i.v. injections of the predetermined
LD50 dose of L-Ann, free Ann, or Dox. The control group was
treated with normal saline. Blood was drawn at 96 h, and the
WBC count, differential, and platelet count were determined.
The experiment was performed twice.
Chronic Lethality and Cardiotoxicity Study. Two
chronic toxicity studies were performed using variations of the
Bentazzoli test (10, 11). In the first experiment, animals (10-
15/group) were treated with weekly iv. injections of 50% of the
predetermined LD10 of L-Ann, free Ann, on Dox. The expeni-
ment was terminated after 6 weekly injections because of sig-
nificant mortality in the group treated with Dox.
In the second experiment, animals (13-15/group) were
treated with 10 weekly iv. doses of 20%, 30%, on 40% of the
predetermined LD10 of L-Ann, free Ann, or Dox. Animals were
weighed weekly, and survival in the different groups was re-
corded.
Animals were killed 1 week after the last drug injection,
and their hearts were removed and placed in formaldehyde.
After fixation, the hearts were dehydrated and embedded in
glycerol methacrybate. Two microsections of the heart were
stained with toluidine blue and examined by light microscopy
for characteristic Dox vacuolization of the myocandial fibers in
a blind fashion. The extent and severity of the lesions were each
scored from 0 to 3 in each individual heart, and a final scone was
calculated as the product of the two scones. Results in a given
group were expressed as the means of all individual scores. The
evaluation was coordinated by Dr. L. C. Stephens in the Section
of Veterinary Pathology at the M. D. Anderson Cancer Center.
Skin Toxicity. The abdominal hair of CD1 mice was
shaved with a hair remover (Carter-Wallace, Inc., New York,
NY), and the exposed abdominal skin was cleaned with warm
water. One day later, groups of 10-11 mice were injected i.d.
with 0.1 ml of a solution containing 1 mg/mb Dox, free Ann, or
L-Ann. Animals were observed daily, and the presence of ery-
thema or ulceration was recorded.
Pathology Studies. CD1 mice (6/group) were given sin-
gle i.v. injections of the predetermined LD50 of L-Ann, free
Ann, on Dox. All mice were killed on day 4, and a complete
histopathobogical study was performed on each.
Dog Toxicity Studies
Facilities and Personnel. Dog toxicity studies were con-
ducted in the Department of Veterinary Medicine at the M. D.
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Table 2 Mouse myebosuppression on day 4”
WBC
(/�.i.) (/p.l)
746 ± 331
156 ± 33-
13 ± 9�
6±3
Platelet(X 103/p.l)
1409 ± 466
1438 ± 565
1593 ± 587
1113 ± 321
Clinical Cancer Research 1371
Table 1 Mouse subacute toxicity”
LD1()5 LD50” LD�)C
(mg/kg) (mg/kg) (mg/kg)
Drug Exp. 1d Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2
Dox 16.4 ± 1.7 14.7 ± 1.1 20.0 ± 2.0 19.6 ± 1.5 24.3 ± 2.5 26.2 ± 2.0
Free Ann 6.0 ± 1.0 7.7 ± 1.2 8.9 ± 1.4 8.8 ± 0.9 13.2 ± 1.0 10.0 ± 2.2
L-Ann 10.1 ± 0.5 10.7 ± 0.7 15.1 ± 2.0 16.4 ± 1.1 22.4 ± 1.9 25.0 ± 1.6
a Seven-week-old male CD1 mice were each given a single iv. bolus injection via the tail vein; 5-7
groups/drug were tested, 10 mice/group.b ,� < 001 between any two drugs.
C � < � between free Ann and Dox or L-Ann; p > 0.05 between Dox and L-Ann.d Exp., experiment.
Anderson Cancer Center in animal facilities accredited by the
American Association for the Accreditation of Laboratory An-
imab Cane. The studies were approved by the Institutional An-
imal Cane and Use Committee.
Dogs were housed in individual indoor runs having a floor
surface of 25 sq ft each. Dogs were fed ad libitum during the
study. They were not i.v. hydrated before drug administration.
Cane of the animals on the study was supervised by Karen J.
Vargas, D.V.M. (Chief, Section of Veterinary Medicine).
Objective and Experimental Design. The objective of
the dog studies was to determine the toxicity of L-Ann in beagle
dogs at 50 and 100% of a dose equivalent to the mouse LD10.
Beagle dogs (two males and two females) age 6 months and
weighing 10-12 kg each were purchased from Marshall Farms
(Northnose, NY). The dogs were administered L-Ann at 50% on
100% of the LD10 (0.71 and 1.42 mg/kg, respectively; one male
and one female/dose) over 15 mm. The dose of L-Ann was
diluted in 50 ml normal saline (final Ann concentration, 0.142-
0.284 mg/mI). The dogs were neither sedated non anesthetized.
They were constantly observed for 8 h after drug administration
and then at least once daily to record any detectable clinical side
effects. Blood counts and chemistries (including bibirubin, cre-
atinine, blood urea nitrogen, serum glutamic-oxaboacetic trans-
aminase, serum glutamic-pyruvic transaminase, alkaline phos-
phatase, lactate dehydnogenase, CPK, total protein, albumin,
sodium, and potassium) were performed twice weekly. All dogs
were killed on day 30, and a complete autopsy was performed
on each.
Dog Pharmacokinetic Studies. Blood samples from the
two dogs treated with 100% of the dose equivalent to the mouse
LD10 (1.42 mg/kg) were obtained at 0, 15, and 30 mm and at 1,
2, 4, 6, 10, and 24 h after infusion. Ann serum concentrations
were determined by HPLC after extraction of serum as previ-
ously described (12). The pharmacokinetic profile was simu-
bated by the Rstnip computer program, and the pharmacokinetic
parameters were calculated.
Statistical Analysis
Differences in toxic doses, peripheral blood counts, and
cardiotoxicity scones among groups of animals were analyzed
for statistical significance using the Student’s t test. Differences
in proportions of animals with skin ulcers were analyzed for
statistical significance using the x2 or Fisher’s exact test as
appropriate.
Control (n = 17) 4600 ± 1600
Dox (a = 12)
Free Ann (n = 1 1)
1100 ± 1200”
600 ± 300”
L-Ann (n = 12) 700 ± 300a Seven-week-old male CD1 mice were each given a single iv.
injection (dose LD50) via the tail vein on day 0. Blood samples were
taken on day 4. Data are mean ± SD.
b ,, > 0� 1 compared with free Ann and L-Ann.
(� p < 0.001 compared with free Ann and L-Ann.d ,, > 005 compared with L-Ann.e ,� < 005 compared with L-Ann.
RESULTS
Mouse Studies
Subacute Toxicity. For the three drugs tested, most
deaths due to the drugs occurred between days 4 and 8. Table 1
shows the results of two separate toxicity experiments. The
mean LD10 and LD50 were 15.6 and 19.9 mg/kg for Dox, 6.8
and 8.8 mg/kg for free Ann, and 10.4 and 15.74 mg/kg for
L-Ann (P < 0.01 between any two drugs). Ann was therefore
about 2-fold more toxic than Dox. The liposome cannier reduced
the toxicity of Ann. As a result, L-Ann was only slightly more
toxic than Dox.
Myebosuppression. Blood counts were determined 4
days after mice were treated with the LD50 of the different
drugs. As shown in Table 2, the granubocyte count was 10-fold
bower in animals treated with free Ann than those treated with
Dox (gnanubocytes/mm3, 13 ± 9 for free Ann, 156 ± 33 for
Dox, and 746 ± 331 for control; P < 0.001 between free Ann
and Dox). The granubocyte count was 2-fold lower in animals
treated with L-Ann than in those treated with free Ann (6 ± 3
versus 13 ± 9, P < 0.05). No significant decreases in platelet
count were observed with any of the three drugs.
Pathology. Mice were killed 4 days after receiving the
LD50 of the different drugs. All mice treated with Dox showed
histopathobogical changes in the kidney (nephrosis), bone mar-
now (aplasia), and gastrointestinal tract (crypt cell necrosis).
Mice treated with L-Ann also showed bone marrow aplasia and
crypt cell necrosis in the gastrointestinal tract, but no kidney
lesions. Free Ann showed a toxicity pattern similar to that of
L-Ann.
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at
C)0
�00
0 14 28 42 56 70
Days
Fig. 2 Animal body weight changes with the chronic administration ofL-Ann and Dox. CD1 mice were administered weekly iv. injections for10 consecutive weeks of 20% (a), 30% (� . . .) or 40% (-) of the LD10of L-Ann (0) on Dox (h). Animals were weighed weekly.
1372 Toxicity of Liposomal Annamycin
Ta ble 3 Skin toxicity”
% Animals with skin damage
(No. of animals)
Day 7 Day 14
Normal Erythema Ulcer Normal Erythema Ulcer
Control
DoxFree AnnL-Ann
100 (10/10)
0 (0/10)10 (1/10)9(1/11)
0 (0/10)
50 (5/10)70 (7/10)82(9/11)
0 (0/10) 100 (10/10)
50 (5/10) 0 (0/10)”20 (2/10) 50 (5/10)’9(1/11) 91 (10/11)
0 (0/10)
50 (5/10)”40 (4/10)’9(1/11)
0 (0/10)
50 (5/10)”10 (1/10)�0(0/11)
a Seven-week-old male CD1 mice were each given an s.c. injection of0.1 ml ofdrug solution or suspension(concentration, 1.0 mg/mb) in the abdominal wall (10-11 mice/group). Control animals were injected with thesame volume of normal saline.
h ,� < 0.025 compared with free Ann and L-Ann.
C � = 0.06 compared with L-Ann.
Table 4 Mouse cardiotoxicity”
% Animals with % Animals
Dose heart lesions aliveDrug (% LD10) Mean score (no.) (no.)
Experiment 1 (6 weekly iv. injections)
Control 0.00 0(0/10) 100 (10/10)Dox 50 287b 100(4/4) 40(4/10)
Free Ann 50 1.50C 25 (2/8) 80 (8/10)L-Ann 50 0.00 0(0/12) 80(12/15)
Experiment 2 (10 weekly iv. injections)
Dox 20 0.50 43(6/14) 93 (14/15)
30 066d 60(9/15) 100(15/15)40 2.00” 100 (5/5) 33 (5/15)
L-Ann 20 0.04 8(1/13) 100 (13/13)30 0.03 7(1/15) 100 (15/15)
40 0.00 0(0/14) 93 (14/15)
a Seven-week-old male CDI mice were each given weekly iv.
injections of 50% of the LD10 of L-Ann, free Ann, or Dox (10 mice!
group) for 6 weeks (experiment 1) or 20%, 30%, or 40% of the LD10 ofthe same drugs (15 mice/group) for 10 weeks (experiment 2). Controlanimals were given injections of the same volume of normal saline.
b� > 0�1 compared with free Ann.C � < ��#{216}5compared with L-Ann.
d � < 005 compared with the same dose fraction of Dox.
Skin Toxicity. Table 3 shows the results of the skin
toxicity experiments. Groups of CD1 mice were given i.d.
injections of 0. 1 ml of a solution containing 1 mg/mb Dox, free
Ann, or L-Ann. Necrotic ulcers were observed in all animals
treated with Dox, basting > 14 days in 50% of them. Necrotic
ulcers were also observed in all mice treated with free Ann, but
these were smaller and resolved much fasten: only 20% and 10%
of mice showed ulcers on days 7 and 14, respectively. Most
mice treated with L-Ann showed only an enythematous reaction;
only 1 of 1 1 showed an ulcer on day 7, which resolved by day
14. L-Ann therefore had a much weaken vesicant effect than
Dox when injected i.d. in the abdominal wall of CD1 mice.
Chronic Lethality and Cardiotoxicity Studies. Table 4
shows the results of the chronic cardiotoxicity studies. Depend-
ing on the dose, drug-induced heart lesions were observed in
43-100% of mice treated with Dox versus only 0-8% of mice
given injections of equitoxic doses of L-Ann. This and the fact
that L-Ann induced bess severe heart lesions (mean scone, 0.00-
0.04 for L-Ann versus 0.5-2.87 for Dox; P < 0.05) indicates
that L-Ann is much less candiotoxic than Dox. The effect of the
different structure of Ann on its candiotoxic potential was stud-
ied in experiment 1. Two (25%) of 8 mice treated with free Ann
that completed the study had cardiac lesions (mean scone, 1.50).
In contrast, all 4 mice (100%) treated with Dox that completed
the study showed severe heart lesions (mean scone, 2.87).
When administered on a weekly schedule, Dox displayed
higher cumulative toxicity, as assessed by a markedly increased
body weight loss at all dose bevels tested (Fig. 2) and the
percentage of animals alive at the completion of the study
(Table 4). Six weekly injections of 50% of the Dox LDIt) caused
60% mortality versus 20% mortality for free Ann and L-Ann.
Ten weekly injections of 40% of the Dox LDJO caused 67%
mortality versus 7% mortality for L-Ann.
Dog Toxicity Studies
Side Effects. Acute side effects were limited to anxiety
and mucosal redness in two of four dogs while the drug was
being administered. These effects may be rebated to a liberation
of histamine or a reaction to being restrained. The reactions
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0 2 4 6 8 10
Hours after admInIstratIon
Clinical Cancer Research 1373
E
C
aC0
0
C
aEC
C
Fig. 3 Ann clearance from plasma of two beagle dogs (#{149},male; 0,female) treated with a dose of L-Ann equivalent to the mouse LD10.
subsided immediately after completion of drug infusion when
dogs were no longer restrained.
No other side effects were observed within 24 h of drug
administration on thereafter. No nausea, vomiting, diarrhea, on
loss of appetite was observed.
Blood Chemistries. Dogs underwent blood counts and
chemistry determinations twice a week for 4 weeks. No signif-
icant changes were observed in any case. One animal did de-
vebop an increased CPK level 2 days after drug infusion, which
corresponded to CPK MM (muscular isoenzyme), thus indicat-
ing that the increase was most likely due to muscular stress
while fighting the restraint during drug administration.
Pathology. All dogs were killed on day 30. No gross on
histopathobogicab changes were observed.
Dog Pharmacokinetic Studies. Fig. 3 shows the plasma
pharmacokinetic profile of L-Ann in two beagle dogs treated
with the mouse-equivalent LD10 during a 15-mm period. Drug
clearance from plasma was found to fit a two-compartment
model with a t112a of 0.41 h and a t112�3 of 2.18 h. The plasma
peak drug level was 1.24 p.g/mb. The area under the curve was
1 .72 �i.g X h/mI. No other fluorescent peaks corresponding to
drug metabolites were detected (12).
DISCUSSION
This study demonstrates important differences in the tox-
icity spectra of L-Ann and Dox and confirms the beneficial
effects of biposomes as carriers of anthracyclines in general. In
addition, the dog studies, which were performed to establish the
starting dose of the current Phase I clinical trial with L-Ann,
indicated that one-tenth of a dose equivalent to the mouse LD10,
the standard starting dose for Phase I studies, should be safe in
humans.
The liposome carrier was shown to reduce the general
toxicity of Ann in mice by increasing the LDS() by 2-fold and
reducing its vesicant activity and candiotoxic potential. Similar
beneficial effects have been previously reported in studies in
which liposomes were used as carriers of the parent compound,
Dox (13-16) encapsulated within the inner aqueous space of
small biposomes (100-200 nm) rather than incorporated within
the liposome membranes of liposomes of similar size as in the
case of Ann. Although a direct comparison of L-Ann with some
of the liposomab Dox formulations currently in clinical trials
might have been of interest, these studies could not be per-
formed, because none of these formulations is commercially
available, and we thought that their preparation in our laboratory
might not result in an identical product. Therefore, although our
studies confirm the beneficial effects of liposome delivery for
anthracyclines in general, no conclusions can be made regarding
which of the currently liposomab anthracycline preparations has
a more favorable toxicity profile. This information will have to
be generated from the ongoing and future clinical trials with
these preparations. No definite conclusions can be drawn re-
garding differences in candiotoxic potential between Dox and
free Ann because of the small number of animals used. How-
ever, Ann by itself seemed to be less cardiotoxic, which is not
surprising in view of the fact that two of the chemical modifi-
cations of Ann have been previously reported to confer a de-
creased cardiotoxic potential (4’-epirubicin and 3’-deamination;
Refs. 17 and 18).
The liposomes not only decrease Ann’s toxicity but have
also been shown to enhance its antitumon activity in vivo in a
variety of tumor models, including mouse lung, liven, and s.c.
tumors as a result of changes in pharmacokinetics and tumor
drug delivery (9, 14, 19, 20). However, the major potential
advantages of L-Ann over Dox and the main justification for its
development are not the beneficial effects provided by the
biposome cannier but its remarkable cytotoxic potential, whether
free or biposome incorporated, against MDR1 tumor cells both
in vitro and in vivo (4). None of the liposomal Dox formulations
currently under active clinical development has been shown to
display in vivo antitumor activity against MDR1 tumors.
Gnanubocytopenia was much more severe with Ann, either
free or biposome incorporated, than with Dox at the LD50 dose
level, which suggests that Ann may be more selective than Dox
for the treatment of bone marrow malignancies such as acute
leukemia. The increased granubocytopenia observed with Ann
was mostly due to its different structure rather than the biposome
carrier. However, granubocytopenia seemed to be somewhat
enhanced by the biposome carrier, probably as a result of en-
hanced delivery of Ann to the bone marrow by the type of
liposomes used. A possible explanation for the increased intnin-
sic bone marrow toxicity of Ann compared with Dox is that
hematopoietic progenitor cells express the MDR1 phenotype
and, therefore, should be more sensitive to agents that circum-
vent MDR1 (21, 22).
At the LD10, L-Ann caused no side effects in beagle dogs,
thus indicating that one-tenth of the LD10, the standard starting
dose for Phase I trials in humans, should be a safe dose. The
peak plasma level and area under the curve at this dose were
1.24 �i.g/ml and 1.7 p.g X h/mI, respectively. No beagle dogs
were given Dox as control to keep to a minimum the number of
animals and because results of similar studies in beagle dogs
have already been published. In a recent study by Gabizon et a!.
(23), beagle dogs were treated with 0.5 mg/kg Dox as an i.v.
bobus (in our case, the dose was 1.42 mg/kg as a 15-mm
infusion); the peak plasma level was about 3 �i.g/ml, and plasma
bevels were below 0.01 p.g/ml at about 30 mm, thus suggesting
a half-life of only a few minutes. In the case of L-Ann, plasma
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1374 Toxicity of Liposomal Annamycin
bevels reached the level of 0.01 jig/mb at 9 h, and the elimination
half-life was about 2 h. Interestingly, no metabolites of Ann
were detected in plasma.
The preclinical studies presented here cleanly indicate that
L-Ann may be more selectively myebotoxic than Dox and may
be noncandiotoxic. These features seem to be mainly the result
of the modulation of Ann’s pharmacology by the liposomes,
although a contribution rebated to Ann’s modified structure
cannot be ruled out. The favorable toxicity profile of L-Ann,
combined with its ability to circumvent MDR1, strongly justi-
fies the introduction of L-Ann in clinical trials. If these proper-
ties are confirmed in human studies, L-Ann may be a useful
agent in the treatment of tumors that express MDR1 at diagnosis
on at relapse. Furthermore, the possibility of giving higher
cumulative doses of L-Ann without candiotoxicity opens the
possibility of giving it in high-dose chemotherapy protocols
followed by bone marrow support. If the initial clinical studies
indicate that L-Ann is a promising new anticancer agent, see-
ond-generation liposomal formulations with enhanced tumor-
targeting properties will be developed.
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