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Experimental Therapeutics |
Pediatric Endocrinology Unit, Karolinska Institute [K. J., K. S., O. S.], and Karolinska Pharmacy [S. E.], Karolinska Hospital, 171 76 Stockholm, Sweden; Department of Pediatrics, University of Turku, 20520 Turku, Finland [K. J., T. T. S.]; and Children’s Hospital, University of Helsinki, 00290 Helsinki, Finland [K. J., T. J.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Amifostine (Ethyol), a recently introduced cytoprotective drug, has been demonstrated to protect normal tissue from the toxic effects of radiation therapy and cytotoxic drugs both in animal models and clinical studies (2, 3, 4, 5) . In vitro, amifostine may even diminish the potential genotoxic and carcinogenic effects of the cancer therapy (2 , 5) . Amifostine is believed to scavenge free radicals, disarm radicals on essential molecules, and form mixed disulfides to protect normal cells (2) . In contrast, malignant cells are not protected, and the enhancement of chemo- and radiotherapy has been observed in clinical studies (6 , 7) . The selectivity, in terms of cytoprotection of normal tissues, is assumed to be related to the perfusion-related distribution and absorption of amifostine and a greater alkaline phosphatase activity in normal than in malignant tissues (2 , 3) .
Knowledge of the usefulness of amifostine in children is poor. It is not known whether amifostine protects against the long-term toxicity of cytostatic drugs and radiation, or whether amifostine itself may have some late effects in children. Only a few clinical trials with amifostine in children have been published, mainly including short-term follow-ups among relapsed poor-risk patients (8, 9, 10) . In these studies, significant protection of amifostine against the early hematological toxicity of chemotherapy has been reported. Doxorubicin is a cytotoxic drug known to bind and intercalate DNA, to inhibit topoisomerase II activity, to generate free radicals, and to covalently bind to DNA after reductive metabolism (11) . Observed side effects in clinical trials and rat model studies included myelosuppression, alopecia, cataract formation, chronic cardiotoxicity, and testicular toxicity (12, 13, 14, 15, 16, 17) . As there are no reports of the effects of amifostine on growing immature individuals, we performed the present study to evaluate the possibility that amifostine would protect against doxorubicin induced toxicity in immature rats. Specifically, we evaluated protection against long-term toxicity and the effects of amifostine on growing immature tissues.
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MATERIALS AND METHODS |
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Treatment Protocol.
The animals were randomized, and 10 in each group were treated at a
postnatal age of 6 days with a single i.p. injection of either 3 mg/kg
doxorubicin, 200 mg/ml amifostine, or saline. Another three groups of
10 animals received an i.p. injection of 50, 100, or 200 mg/kg
amifostine, respectively, as a pretreatment 15 min before 3 mg/kg of
doxorubicin i.p. Amifostine (Schering Plough, Stockholm, Sweden) and
lyophilized doxorubicin (Pharmacia Upjohn, Stockholm, Sweden) were
dissolved in saline immediately before use and injected in dose volumes
of 0.06–0.25 ml/100 g of body weight (50 mg/ml) and 0.15 ml/100 g of
body weight (2 mg/ml), respectively. The body temperature was measured
in control, amifostine only, doxorubicin only, and doxorubicin and
amifostine 50 mg/kg pretreated animals at 0, 30, 60, and 90 min and at
20 h after the first injection. A small but statistically
insignificant decrease in body temperature was observed 60 min after
the first injection of amifostine in each treatment group (temperature,
0 min and 60 min: control, 33.3 ± 0.2 and
32.7 ± 0.2°C; amifostine 200 mg/kg, 33.4 ± 0.1 and 31.7 ± 0.2°C; doxorubicin 3 mg/kg,
32.7 ± 0.2 and 32.6 ± 0.2°C; and
amifostine 50 mg/kg + doxorubicin 3 mg/kg, 33.7 ± 0.1 and 33.0 ± 0.2°C, respectively). Body
temperature normalized to the pretreatment level in all groups within
90 min after treatment and was stabile for the next 20 h of
follow-up. No statistically significant differences in body temperature
were seen between the different treatment groups.
In a second experiment, 20 young rats at the age of 6 days were treated by injection of 3 mg/kg doxorubicin i.v. in the tail vein. Ten of these animals were treated with 50 mg/kg amifostine i.p. 15 min before doxorubicin administration.
Study Variables.
Animals were observed and weighed weekly, and the appearance of
alopecia and cataract as well as the deaths were noted. The study was
terminated when animals were 80 days of age, i.e., 74
days after the start of the treatment. Animals were euthanized with an
overdose of carbon dioxide, and blood samples were collected for
testosterone and leptin analyses. Body weight and nose-tail length were
measured. The heart, spleen, liver, right kidney, right testis, right
epididymis, and right seminal vesicle were excised from each animal and
weighed. Hearts were immersion-fixed in 10% neutral buffered formalin
and testes were immersion-fixed in Bouin’s fixative. The
samples were embedded in paraffin and prepared for light microscopic
examination.
Testosterone and Leptin Analyses.
Testosterone and leptin were assayed in plasma samples. Testosterone
was measured by a Coat-a-Count radioimmunoassay kit (Diagnostic
Products Corporation, Los Angeles, CA), and leptin was measured by a
rat-specific radioimmunoassay kit (Diagnostika, Stockholm, Sweden)
according to the manufacturers’ instructions.
Statistical Analysis.
Data are presented as the mean values ± SE. The
Mann-Whitney nonparametric U test was used for
comparison of two independent sample populations. Kruskall-Wallis
analyses with a Dunn’s posttest were performed for
multiple comparison of independent sample populations. The mortality
data were analyzed using the Kaplan-Meier method with a log-rank test
for the comparison of curves. A P < 0.05 was
considered significant.
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RESULTS |
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The eyes of the animals were opened during the 2nd week after treatment
and were judged to be normal in all treatment groups. The incidence of
cataract increased slowly during the 3rd week after doxorubicin
treatment, reaching its maximum (70% of all living animals) 25 days
after doxorubicin injection (Fig. 1A)
. Pretreatment with 50 mg/kg of amifostine before
doxorubicin significantly decreased cataract formation. Only 10% of
all living animals in this treatment group had developed cataract 25
days after treatment (Fig. 1A)
. Animals receiving the higher
amifostine doses (100 mg/kg and 200 mg/kg) showed a rapid appearance of
cataract during the 2nd week after treatment. The incidence of cataract
in these treatment groups was already 70% of all living animals
18 days after the treatment (Fig. 1A)
. No cataract was
observed in control or amifostine-only groups.
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. Altogether, 90% of living
animals had lost their neck hair during the 2nd week after treatment.
Thereafter, regrowth of the fur was observed, and alopecia disappeared.
In contrast, amifostine-pretreated animals did not lose their neck hair
during the 1st and 2nd weeks after doxorubicin treatment. All
amifostine doses were effective to protect against alopecia (Fig. 1B)
. A small injection-site alopecia not affected by
amifostine pretreatment was found in all doxorubicin-treated rats. No
alopecia was observed in control or amifostine-only-treated animals.
Mortality after Doxorubicin and Amifostine Pretreatment.
Data on mortality during the course of study are shown in Fig. 2
. A statistically significant (P = 0.015)
difference in survival between the different treatment groups was
observed. Of the doxorubicin-only-treated animals, 3 of 10 died before
termination of the experiment, giving a survival rate of 70%. Two of
the deaths occurred during the 2nd week after treatment, and the third
death occurred 65 days after treatment. There was no early mortality in
the animal group pretreated with 50 mg/kg amifostine. However, 4 of 10
animals died after 45 days of follow-up, giving a survival rate of
60%. Pretreatment with 100 mg/kg and 200 mg/kg of amifostine was toxic
to young rats. The mortality in these groups started to increase after
45 days of follow-up and reached 80% at the time of cessation of
experiment when the animals were 80 days of age. At the necropsy, the
most consistent gross pathological changes found in animals with early
deaths were excessive amounts of peritoneal fluid and a pale
discoloration of the kidneys and the intestine. Organ weights of three
rapidly necropsied 200 mg/kg amifostine-pretreated rats were obtained
at the ages of 52, 65, and 66 days. Body weights of these animals were
100 g, 196 g, and 224 g; heart weights were 642 mg, 761
mg, and 1196 mg; and testis weights were 210 mg, 280 mg, and 151 mg,
respectively. There were no deaths in the group of 10 control animals.
In the amifostine-only treated group, one animal was lost during the
1st day of the experiment because of bleeding after injection.
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. Because animals receiving doxorubicin were so much smaller,
additional comparison of organ weights was made with relative index
values (organ weight/body weight). In the doxorubicin-treated group,
heart weight indices were significantly increased and testicular,
epididymis, and seminal vesicle weight indices significantly decreased
as compared with control and amifostine-only-treated groups (Table 1)
.
The organ weight indices in the amifostine-pretreated group did not
differ from those in the doxorubicin-only treated group (Table 1)
.
There were no changes in weight indices of other investigated organs
after doxorubicin treatment compared with control, amifostine-only, or
amifostine-pretreatment groups (Table 1)
. Body and organ weights in
amifostine-only-treated groups were comparable with controls (Table 1)
.
At necropsy, the gross pathological changes in doxorubicin-treated and
amifostine-pretreated animals were comparable with those seen in the
animals with early deaths. No gross abnormalities were observed in
animals given amifostine-only or in saline-treated controls. The high
mortality in the 100 mg/kg and 200 mg/kg amifostine-pretreated groups
did not allow additional analysis of any protective effect at the end
of experiment. The mean index values for heart (mg/g index,
4.8 ± 1.5) and testis (mg/g index, 1.5 ± 0.9) weights were measured for three necropsied (dead 14, 15,
and 28 days before cessation of experiment) and two surviving rats in
the 200 mg/kg amifostine-pretreatment group. No statistical difference
(P > 0.05) was observed when these values were compared
with the mean index values for heart (mg/g index, 4.5 ± 0.2) and testis (mg/g index, 1.2 ± 0.2) in the
doxorubicin-treated group at the end of experiment.
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Histological Morphology of Cardiac and Testicular Tissue.
Testicular tissue of animals treated with a single dose of doxorubicin
i.p. at the age of 6 days showed major damage of the spermatogenic
epithelium at the age of 80 days. Sertoli-cell-only tubules with some
degenerating spermatogenic cells were seen in histological sections.
Leydig cell hyperplasia was observed in the interstitium. The damage in
testicular morphology after amifostine 50 mg/kg pretreatment was
similar to that after doxorubicin-only treatment, and no signs of
protection were seen at the microscopic level. Control animals and
amifostine-only-treated animals showed normal testicular morphology. In
light microscopic sections from myocardium of doxorubicin i.p.-treated
animals, the alterations consistent with doxorubicin-induced
toxicity were seen. Cardiomyocytes were vacuolated, and edema of
the cells was observed. Similar changes were observed also after
amifostine 50 mg/kg pretreatment. Control animals and
amifostine-only-treated animals showed normal myocardial morphology.
Testosterone and Leptin Levels after Amifostine and Doxorubicin
Treatment.
Testosterone levels in control, amifostine-only, doxorubicin i.p.-only,
and amifostine 50 mg/kg-pretreated doxorubicin i.p. groups were
4.1 ± 0.9 ng/l, 1.6 ± 0.6 ng/l,
3.1 ± 1.1 ng/l, and 5.8 ± 2.1 ng/l,
respectively, at the end of the experiment. There was no statistically
significant difference between the treatment groups. Leptin levels in
the same treatment groups were 5.7 ± 0.7 ng/l,
7.7 ± 1.1 ng/l, 3,7 ± 1.1 ng/l, and
3.7 ± 1.0 ng/l, respectively. There was a statistically
significant difference (P < 0.05) in leptin levels between
amifostine-only- and doxorubicin-only-treated groups and
amifostine-only and amifostine-pretreated groups.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A high rate of cataract formation is reported after pediatric bone marrow transplantation (18 , 19) . The growing lens during infancy is known to be sensitive to irradiation, high-dose chemotherapy, and systemic steroids (14 , 18) . Busulfan, a cytotoxic drug, has been shown to induce a reduction of mitotic activity in the lens epithelium and nuclear disintegration with chromatin aggregation leading to cataract formation in rats and humans (20 , 21) . In the present study, a low dose 50 mg/kg of amifostine showed significant protection against the doxorubicin-induced changes in the lens and decreased cataract formation.
Earlier clinical studies have suggested that young children may tolerate higher amifostine doses than adult patients, and that higher doses of amifostine in children give increased protection against myelosuppression (8) . According to these results, an amifostine dose of 1650 mg/m2 has been recommended for future Phase II pediatric trials. The recommended dose in adults for myeloprotection is 740–910 mg/m2 (4 , 5) . In light of these recommendations, the present observations that high doses of amifostine (100–200 mg/kg equal to 300–600 mg/m2; Ref. 22 ) in young growing rats actually increased late toxicity of doxorubicin and caused late mortality, are worrisome. An amifostine dose of 200 mg/kg has been reported to protect various tissues against doxorubicin toxicity in adult rats (3) . In the present study, the young rats initially tolerated high amifostine doses, and there was protection against early toxicity of doxorubicin with these doses. Cataract appeared as the first sign of the adverse effects of high amifostine doses, and mortality of the animals increased markedly after 45 days of follow-up. At necropsy, the gross pathological changes compatible with doxorubicin toxicity were seen in animals with early deaths. A similarly increased late mortality 9–11 weeks after the use of amifostine and repeated doses of doxorubicin has been reported recently in spontaneous hypertensive rats (23) . Our results show the importance of long-term follow-up when optimal doses of amifostine are selected for the treatment of children.
In the present immature rat model, the best protection against alopecia and cataract formation was reached with a dose of 50 mg/kg of amifostine. With this dose, long-term survival was comparable with the doxorubicin-treated group. However, when the effects of doxorubicin in heart and testicular spermatogenic epithelia were evaluated, no significant protection was seen. The animals grew poorly in both amifostine-pretreated and doxorubicin-only-treated groups. The present study shows clearly that amifostine does not have protective effect against the late toxicity of doxorubicin when the lower doses with acceptable mortality were used. Our results from rats with early deaths, receiving the highest dose of amifostine, do not suggest that an increased dose of amifostine has protective effect in the sensitive organs. Among some laboratory animal strains, extreme reduction in body temperature after treatment with amifostine has been reported as leading to peripheral vasoconstriction, decreased renal clearance, and potentiation of chemotherapy toxicity (24) . We could not detect any significant changes in body temperature after amifostine injection, suggesting that changes in body temperature have not interfered with the results in the presently used animal model.
In the present study, marked toxicity of a single i.p. injection of doxorubicin in young growing animals was detected. The degree of weight loss was more pronounced than the decrease in linear growth. The decrease in plasma levels of leptin in doxorubicin-treated groups also suggests a decrease in fat mass, as leptin concentration in blood is known to be proportional to body fat mass, both in humans and rodents (25) . Gastrointestinal lesions caused by the local effects of i.p. injections might have been an important factor in the decline in body weight. However, the i.p. route of doxorubicin was not shown to create increased lesions in intraabdominal organs or to affect the amifostine protection. When the study was repeated with doses given i.v., a similar decline in body weight was seen, and the gross intestinal pathology in necropsy was comparable with i.p.-treated animals. No protection with amifostine was seen, irrespective of the doxorubicin administration route chosen.
The observed tissue necrosis at the injection site of the tail neck in the treated group is obviously attributable to some tissue leakage of doxorubicin. This may affect the pharmacokinetics of doxorubicin and decrease the peak plasma levels of the drug. The observed slightly lower mortality, lower incidence of alopecia, and decreased testicular toxicity in the i.v.- compared with the i.p.-treated group may be a reflection of this phenomena. Interestingly, the i.v. route of administration of doxorubicin abolished the protective effect of 50 mg/kg amifostine on cataract formation. This suggests that the timing of amifostine dosing to reach protective concentrations in correlation to peak plasma levels of doxorubicin may be critical for ocular protection. Another possibility behind the higher testicular toxicity observed in the i.p.-treated group is that doxorubicin may have a direct toxic effect in the testicular tissue by diffusion through the testicular capsule. This aspect is important when planning additional studies on testicular toxicity in very immature animals. In contrast, the heart was damaged both with i.p. and i.v. injections of doxorubicin. This suggests that the heart in young rats is very sensitive to doxorubicin, which is in agreement with clinical observations that the risk of long-term cardiotoxicity is increased when treatment is given to very young children (26) .
In conclusion, our results demonstrate that amifostine has a significant protective effect against early toxicity of doxorubicin in young growing animals. However, amifostine did not protect young rats against late toxic effects of doxorubicin on linear growth, heart, or testicular tissue. More worrisome, the mortality was actually found to be increased after pretreatment with high doses of amifostine. Additional studies analyzing the safety of amifostine treatment and the mechanisms of exacerbation of doxorubicin toxicity by amifostine in young growing animals are highly warranted before a wider use of this drug in pediatric cancer patients can be recommended.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Swedish Children’s
Cancer Fund, the Finnish Cancer Society, the Finnish Cultural
Foundation, and the Finnish Pediatric Research Foundation; from the
Nona and Kullervo Väre Foundation (to K. J.); from the Åke
Wiberg Foundation, the Cancer and Traffic Injury Victims Foundation,
the Cancer and Allergy Fund; and by Grants 8282 and 11412 from the
Swedish Medical Research Council (to O. S.). 
2 To whom requests for reprints should be
addressed, at Children’s Hospital, University of Helsinki,
Stenbäckinkatu 11, FIN-00290 Helsinki, Finland. Phone:
358-9-4711; Fax: 358-9-47174707; E-mail: Kirsi.Jahnukainen{at}hus.fi
Received 1/15/01. Accepted 7/ 3/01.
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REFERENCES |
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