preclinical toxicity of liposome-incorporated annamycin ... · chronic lethality and cardiotoxicity...

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.

Association for Cancer Research. by guest on August 26, 2020. Copyright 1995 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

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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.



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.



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.


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



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




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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preclinical toxicity of liposome-incorporated annamycin ... · chronic lethality and cardiotoxicity...

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