Antitumor effect and peritumoral brain edema formation in relation to MX2, ACNU, and doxorubicin therapy: A comparative analysis using rodent models of gliomas
Anthracyclines such as doxorubicin (DOX) are important chemotherapeutic agents because of their powerful antitumor effects on a wide range of human malignant neoplasm”. However, DOX and other anthracyclines have seldom been utilized in the treatment of malignant gliomas because their anti-glioma effects are not potent enough, probably because they do not cross the bloodbrain barrier . Particularly in the case of DOX, there is a dose-dependent cardiotoxicity that may limit the clinical utility’s$
MX2,3′-Beam ino-3′-morphol ino-13-deoxo-10-hydroxycarminomycin hydrochloride, which is a new morpholino anthracycline and a derivative of DOX, has been reported to penetrate the blood-brain barrier (BBB) significantly9′ 10 and its toxicity appears to be much lower than that of DOX”. Some studies have reported that MX2 inhibited glioma growth in vitro , and in vivo14, however, the data is lacking regarding antiglioma effect of MX2 in comparison with other chemotherapeutic agents commonly used in the clinical setting. This comparison in vivo seems to be very important, since the clinical effectiveness of MX2 is not yet fully examined and a phase II study of MX2 in patients with recurrent malignant glioma has just been recently published”.
ACNU, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3(2-chloroethyl)-3-nitrosourea hydrochloride, has been evaluated to be one of the most potent drugs in the glioma therapy. In contrast, DOX, which is one of the most popular drugs against neoplasm, is seldom used against glioma. As objects to compare with MX2 in this study, therefore, we selected two chemotherapeutic agents: (1) ACNU as one of the most commonly used anti-glioma drugs, and (2) DOX in order to test how much the anti-glioma effect of MX2, a derivative of DOX, is augmented. Using rodent glioma models, we examined tumor-growth inhibition, survival rate, and severity of peritumoral brain edema following MX2, ACNU, or DOX therapy.
MATERIALS AND METHODS
All experimental procedures were conducted in accordance with the guidelines for the care and use of laboratory animals in Kochi Medical School, Kochi, Japan.
In each experiment, the same dose and method of drug administration was used: Saline as control, ACNU (30 mg kg-‘), doxorubicin (3 mg kg-1), or MX2 (3 mg kg-1), were injected intraperitoneally (i.p.) once.
Cell lines and implantation
Cells lines of 203 glioma (the mouse origin)16 or 9L glioma (the rat origin)1-7 were used in the following two experiments. In the first, 2x 106 cells of the 203 glioma (0.1 ml volume) were transplanted subcutaneously into the axillary region of the C57 BL/6 mouse weighing 2030 g (SLC Inc., Japan). In the second, 2 x 106 cells of the 9L glioma (101) were injected with a Hamilton syringe into the right striatum of Fisher 344 rats weighing 300350 g (Charles River, Japan). Light ether anesthesia was used for the subcutaneous implantation and pentobarbital anesthesia (50 mg kg-‘ i.p.) was used for the intracerebral implantation.
The day of glioma cell implantation was designated as Day 0.
Tumor growth inhibition study (subcutaneous 203 glioma model)
The mouse subcutaneous glioma model was used to measure drug effects on tumor growth inhibition. Fourteen days after subcutaneous implantation of the 203 glioma (Day 14), diameters of long axis (length) and short axis (width) of the tumor mass palpable from the surface of the skin were measured with a slide caliber. Approximate estimation of tumor volume was calculated according to the formula previously described 16. Tumor volume= lengthx(width) x0.5. The animals were then treated with ACNU (n=25), DOX (n=10), or MX2 (n = 14). One group of animals received saline as control (n=7). On Day 28, the tumor volume was again measured through the skin, the tumor removed under pentobarbital anesthesia (50 mg kg-‘ i.pJ and weighed. Tumor growth (%) in each animal was expressed by (tumor volume on Day 28)/(tumor volume on Day 14) x 100. The tumor growth inhibition (%) by a specific drug was expressed by (11 – T/C) x 100, where T and C is the mean value of the tumor growth calculated in the drug-treated and control groups, respectively. Studies of body weight changes (Day 0, 14, and 28) and white blood cell (WBC) counts in the blood on Day 28 were also addressed in this experiment.
Survival study (intracerebral 9L glioma model)
A total of 79 rats were used for intracerebral implantation of the 9L glioma. Since 19 rats (24%) had died before starting treatment on Day 7, the remaining 60 rats were assigned to four groups: control (n = 16), ACNU (n=14), DOX (n=15), and MX2 (n=15). The drug was administered only on Day 7. Activity of the rats was checked every 8 h. If a rat was found to be severely ill, a more frequent observation was done so that the brain could be removed immediatley after death.
Brain edema study (intracerebral 9L glioma model)
The 60 rats that survived through Day 7 in the 9L glioma model were used for measurement of brain water content with a drying-weighing method. When the rats were found dead before Day 14 (n=33), the whole brains were removed as soon as possible after death and subjected to water content analysis. The brains of the 27 survived rats were removed under pentobarbital anesthesia (50 mg kg-1 i.p.) on Day 14. The cerebellum and the brain stem, and the tumor in the cerebrum were removed from the whole brain, and the rest of the brain split into two cerebral hemispheres. After measurement of the wet weight of the right (tumor side) and the left (non-tumor side) cerebral hemispheres, each was dried in a gravity oven set at 105 deg C for 96 h to determine the dry weight. The brain water content (%) was calculated as follows: (wet weight -dry weight)/wet weight x 100. Water content of the cerebral hemispheres in normal rats without tumor implantation were also measured seven days after injection of a single dose of saline (n = 10), ACNU (n = 8), DOX (n = 8), or MX2 (n = 8). Histological examination
Rats different from those used in the survival study or water content study were separately prepared for histological examination (n=3 in each treatment group). After transcardial perfusion with 10% formalin, the whole brain with tumor was dipped in the same fixative and processed for making 7 (mu)m thick paraffin sections stained with hematoxylin-eosin.
The animal survival study was analyzed by KaplanMeier survival expectancy method (Mantel-Cox). Data of tumor growth inhibition and brain water content were analyzed by ANOVA for statistical significance. Fisher PLSD was used for comparison between two groups only when there was statistical significance among multiple groups. Statistically significant values were determined at p
Tumor growth inhibition
In the mouse 203 glioma model, ACNU prominently inhibited the tumor volume growth (p
Body weight change
Figure 2 shows body weight changes in the mice bearing 203 glioma subcutaneously. The mice in all the groups gained weight equally by about 1 g from Day 0 to Day 14. After receiving the drug, the whole body weight including the tumor mass significantly decreased in ACNU and MX2 treatment groups. Control animal lost the net body weight on Day 28 although increase of body weight was superficially observed because of the enlarged glioma. In contrast, the net body weight loss on Day 28 was minimal in DOX group.
White blood cell counts
Number of WBC in the blood significantly decreased 14 days after administration of any of the three drugs in the 203 glioma model. This adverse effect was most severe in DOX, next in ACNU, and the least in MX2 group (Figure 3).
In the experiment using rat 9L glioma model, the mortality in each treatment group was as follows: 62.5% in control, 43.9% in ACNU, 55.0% in DOX, and 53.3% in MX2 (Figure 4). None of the drugs attenuated mortality (p=0.65 by Mantel-Cox), although only ACNU showed a tendency of amelioration.
Brain edema study
In the rats survived through Day 14 in the intracerebral 9L glioma model, DOX and MX2 compared to control, significantly increased the water content of the cerebral hemispheres not only on the tumor side but also on the non-tumor side (Figure 5). ACNU showed the same tendency but the data did not reach to statistical significance. In contrast, in the normal brain without glioma implantation, ACNU, MX2 and DOX did not cause a statistically significant increase of brain water content compared to control.
Similar to the results in the survived rats, in the rats dead by Day 14, DOX and MX2 with statistical significance and ACNU with tendency, increased water content of the cerebral hemisphere on the tumor side. However, on the non-tumor side, the increase of brain water content did not differ among the four groups. When we compare the brain water content between the survived and the dead rats in the same treatment group, all the groups including control showed that water content on the non-tumor side were significantly higher in the Beat rat brains than in the survived rat brains. However, this significant difference between the dead and the survived rat brains was not observed on the tumor side.
Histological analysis with light microscopy demonstrates that aggravation of brain edema is most severe in MX2, and less in ACNU and DOX treatment groups (Figure 6). In no treatment does the histological finding indicate only a slight degree of peritumoral brain edema.
MX2, a new morpholino anthracycline, has been introduced as a potential antiglioma agent due to its highly lipophilic nature enabling it to easily penetrate the BBB . Since its discovery, MX2 has shown antiglioma properties in experimental studies 12,13,17 . Before going to clinical application of a new agent, a comparison is required with other clinically used agents particularly to their therapeutic potency and toxicity. However, to our knowledge, none of the previous studies have demonstrated comparison between MX2 and other anti-glioma drugs.
In this study, the results in the subcutaneous 203 glioma model clearly demonstrated that MX2 but not DOX inhibited tumor growth significantly, although ACNU was much more potent than MX2. Since this model has no barrier such as the BBB for the drugs to reach the glioma, the anti-glioma effect of MX2 is not merely due to the drug property of penetrating the BBB. Regarding the adverse effect of decrease of WBC, MX2 was the least hazardous among the three tested drugs and particularly much weaker than DOX. The changes of net body weight and tumor weight has indicated the following: (1) In control, the enlargement of tumor severely disturbed natural course of the body weight gain; (2) ACNU and MX2 suppressed growth of the tumor as well as the body itself; and (3) DOX did not decrease the net body weight despite having a mild effect of suppressing the tumor growth. These results suggest that ACNU has the most potent effect but MX2 can be an option for chemotherapy of malignant gliomas.
In the experiment using the intracerebral 9L glioma model, the result that 24% of the rats bearing 9L glioma died even before drug administration suggests that our experimental protocol may have induced very severe brain condition with increased intracranial pressure due to the tumor mass and/or the peritumoral edema. The results of ineffectiveness of ACNU or MX2 shown in the intracerebral 9L glioma model, although significantly effective against the subcutaneous 203 glioma, might have been due to our experimental protocol making therapeutic windows narrowed. In fact, in the previous report” demonstrating the effectiveness of MX2 on 9L glioma in vivo, the authors used the experimental protocol injecting the drug much earlier.
However, interesting results have been derived from our experimental paradigms that have probably induced severely ill condition in the brain. in the survived rats, the brain edema in the cerebral hemisphere on the tumor side as well as on the non-tumor side was aggravated by the chemotherapeutic agents, particularly DOX and MX2. To our knowledge no studies, except those using the method of intra-arterial administrations, have reported that the chemotherapeutic agents aggravated brain edema in the patients with glioma. In the clinical setting, DOX has probably been seldom used against glioma and combined treatments with corticosteroid or hyperosmotic mannitol may mitigate aggravation of brain edema. However, even ACNU has the possibility of accelerating brain edema formation and we need to be more cautious when we start chemotherapy particularly when the tumor is relatively large or the peritumoral edema is prominent. In contrast, the brain edema in the non-tumor side of the cerebral hemisphere was also aggravated by these three drugs. These results may suggest that the non-tumor side of the brain may have the subclinically ill conditions, such as slightly increased intracranial pressure and increased permeability of the BBB, that may be one of the important backgrounds for the chemotherapeutic drugs to induce severe brain edema.
In the dead rats, the same phenomena of brain edema aggravation by the drugs were found only in the tumorside brain. Together with the results that brain edema on the non-tumor side in the dead rats did not differ among the four groups, one of the death-determining factors may be the brain condition contralateral to the tumor side. Even though the brain edema is severe on the ipsilateral side of the tumor, the rats might survive as long as the brain water content on the contralateral hemisphere is below a certain level of the threshold, probably about 79.5% or more.
Dose-dependent bone marrow suppression and neurotoxicity often limits the further use of ACNU despite its potent effect on glioma’ . It has been reported that intra-arterial injection of ACNU caused brain edema in clinical conditions”. In this study, we also found a significant elevation of brain water content in ipsilateral and contralateral sides of the tumor when animals received a single dose of ACNU at a dose of 30 mg kg-1, i.p. This result may suggest that ACNU produces brain edema in clinical standard dose if it is carefully monitored. MX2 has been reported to have lower toxicity than its parent compound DOX . Although a previous study 14 demonstrated the dosedependent increase of concentration of MX2 in the normal brain, the effect of MX2 on brain edema formation was not clarified. In this study, we found that the dose of MX2 (3 mg kg-1, i.p.) caused significant elevation of brain water content both ipsilateral and contralateral to the tumor sides. Our results also demonstrated that ACNU caused less brain edema formation than MX2, therefore, we may suggest that ACNU is better than MX2 in the glioma chemotherapy.
Although those antiglioma agents caused brain edema in the tumor bearing animals, they did not have any significant effect on brain water content in the normal brain. There are two possibilities of increased brain edema formation in the tumor bearing brain; firstly, the increased concentration of the drug in the tumor side, because brain tumor itself causes breakdown of the blood-brain barrier3-6; secondly, the high intracranial pressure due to tumor may contribute to increased brain edema formation. The second may be the most likely reason for aggravating brain water content in contralateral sides. In this study, rat survival rates were not improved with ACNU or MX2 treatment, however, a drug-induced decrease in mortality might occur if the treatment was started earlier or if the post-treatment observation was extended. Further studies are essential to assess and compare the survival capabilities of MX2 and ACNU.
We suggest that MX2 is a reasonable candidate for a chemotherapeutic agent in the treatment of gliomas. However, considering their relative potencies in tumor growth inhibition and drug related brain edema formation ACNU may still be the better choice for glioma chemotherapy. The management of brain edema, however, still remains an issue with not only ACNU and DOX, but with MX2 as well, and is an important concern when selecting chemotherapeutic agents in the treatment of glioma.
Copyright Forefront Publishing Group Dec 2000
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