TOXICOLOGY

AND APPLIEDPHARMACOLOGY33,484-497(1975)

Toxicological and Pharmacological Studies 9-Hydroxy-Ellipticine in Mice

on

S. CROS,~ R. SORBARA,~ CH. MOISAND,~ N. DAT-XUONG,~ P. LECOINTE,’ AND C. PAOLETTI’ Laboratoire de Pharmacologic et Toxicologic Fondamentales du C.N.R.S., 31078 Toulouse Cedex; Laboratoire d’dnatomie Pathologique, U.E.R. Mddecine Purpan, 31300 Toulouse et Institut de Chimie des Substances Natwelles du C.N.R.S., 91190 Gif-sur- Yvette, France Received January 6,1975; accepted May 6,1975

Toxicological and Pharmacological Studies on 9-Hydroxy-Ellipticine in Mice. CROS,S., SORBARA, R., MOISAND, CH., DAT-XUONG, N., LECOINTE, P., AND PAOLETTI, C. (1975). Toxicol. Appl. Pharmacol. 33, 484497. A toxicological study of 9-hydroxy-ellipticine (g-OH-E), a new potent anticancer agent, wascarried out on DBA/Z mice. The drug is not toxic by the po route. By the iv route, it is notably lesstoxic than by the ip route. The toxicity of the product beingcumulative, it hasbeendetermined how many times and at what frequency therapeutic

doses of drug (L1210

leukemia)could be administered.The main signsof acute and subacute toxicity were: weight loss,reversiblehypothermia causedby dosesaslow as one-fifth of the sublethal dose, hematologic changesmainly affecting leukocytes,and neurologicdisorders.The thresholddosefor thesechanges was determined. The number of polynuclear neutrophils decreased rapidly, but quickly returned to normal values,while the lymphocyte count exhibited a slower but more extended reduction. Dosescausingdeath in 24 hr resultedin locomotor disturbancesand paraplegiadue to preferential lesionsof Purkinje cellsin the cerebellum,detectedby light and electron microscopy. Derivatives belonging to the series of ellipticines are endowed with an anticancer action on experimental (Dalton et aE., 1967) and human tumors (Mathe et al,, 1970). New compounds in this serieswere synthesized and submitted to in vitro screening. A correlation was established between their affinity for DNA, into which they intercalate (Festy et al., 1971) and their biological activity (Le Pecq et al., 1974a, b). Along this line, a new compound, 9-hydroxy-ellipticine (9-OH-E) was shown to be as active on experimental L1210 leukemia as were currently used anticancer chemical agents (Le Pecq et al., 1973). It was therefore considered as worthy of clinical trials. Thus a careful toxicological investigation was necessary. This paper describesthe results obtained in mice. Preliminary results in mice and dogs have already been reported (Paoletti et al., 1974; Chanh et al., 1974). 1 Laboratoire de Pharmacologic et Toxicologic Fondamentales. 2 Laboratoire d’Anatomie Pathologique. 3 Institut de Chimie des Substances Naturelles. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

484

TOXICITY

OF %HYDROXY-ELLIPTICINE

485

IN MICE

METHODS

9-Hydroxy-ellipticine (9-OH-E) was synthesized in our laboratory by demethylation in refluxing pyridinium hydrochloride of 9-methoxyellipticine, the latter being obtained from the corresponding 5-methoxy indole derivative (Dalton et al., 1967). The structural formula is shown in Fig. 1.

FIG.

1. 9-Hydroxy-5,l

I-dimethyl-[6H]-pyrido[4,3-blcarbazole.

C,,H,,N,O

= 262.

The hydrochloride crystallizes with one-half molecule of water. Analytical results ( %) for C, H, N, Cl, 0 were 66.61, 5.12, 9.35, 11.81, and 7.74, respectively (observed), compared to 66.72, 4.94, 9.16, 11.60, and 7.84, respectively (expected). As a base, 9-OH-E can be identified by thin-layer chromatography on silica gel, using Schlecher and Schiill plastic plates or on alumina T, using Merck aluminum plates. In the first system, a mixture of ethylacetate : ethanol : 27 % ammonia hydroxide (140 : 70 : 10) was used. In the second one, the solvent was benzene : chloroform : ethanol (90: 60: 30). Spotting was carried out by fluorescence at 366 or 254 nm. R, values were 0.80 and 0.37, respectively. Most of our tests were carried out at final concentrations of 0.5 to 5 mg/ml in 0.1 M acetate buffer, pH 5.0. Under such conditions, absorption was maximum at 305 nm (E 1 %/lcm = 1180). The product can be maintained in the dark, in the cold, for several weeks. Its stability was checked by thin-layer chromatography and uv-spectrophotometry. Its limit of solubility in buffer was 5 mg/ml; when higher concentrations were necessary the drug was suspended in 65 mg/ml gum arabic. Toxicological studies were performed on male and female DBA/2 mice,4 7 wk old, weighing about 20 g, maintained in rooms at 23”C, and fed pellet? with water ad libitum. We also used mice whose phenotype is identical to that of the nervous strain (nr) previously described by Sidman and Green (1970) and Landis (1973). Their neurological behavior was compared with that of the animals treated with 9-OH-E by different tests: fixed rod, rotating rod (15 rot/min, for I min), righting reflex (somersault test) (Turner, 1965). The acute toxicity of 9-OH-E was determined after one injection of material given either in gum arabic by gastric intubation (PO) or in buffer by the intraperitoneal (ip) and intravenous (iv) routes. For 20-g mice, the largest volumes were 1 ml (po-ip) and 0.6 ml (iv). Animals were assigned to groups of ten for each separate dose: five males and five females caged five per cage. LDO and LDlOO (except by the po route) were estimated and the intermediate doses were calculated with logarithmic intervals within the dose-ranges of 45-50 to 250 mg/kg (ip, males or females) and 50-150 mg,‘kg (iv). After treatment the mice were weighed regularly and signs were observed over a period 4 Obtained from the Centre de SBlection des Animaux de Laboratoire France. ’ JOF Souris entretien.

du C.N.R.S. at Gif-sur-Yvette,

486

CROS

ET AL.

of 1 mo. Lethal doses were calculated after 24 hr and at the time of crisis, i.e., the time after which no further death occurred. In some mice, rectal temperature and hematologic values were determined and histologic examinations were carried out. Rectal temperature was registered with a thermoelectric couple (Elektrolaboratoriet, type TE3). Temperature variations were tested by the paired t test (Schwartz, 1963) and for each period of time the activity of the product was estimated by the two-way analysis of variance and Dunnett’s test, to compare treated animals with controls (Grimm, 1973). Hematologic examinations were carried out by classical methods (Bessis, 1954; Ganter and Jolles, 1969). Blood was collected from the caudal vein. Only the percentages of polynuclear neutrophils and of lymphocytes, which account for approximately 98 % of leukocytes, will be reported here. For each animal and for each period of time, the total number of leukocytes per mm3 and the percentages of neutrophils and lymphocytes were measured. On the basis of these data, the total number of neutrophils and lymphocytes was calculated. The results were expressed as the percent variations of these values compared with pretreatment values. Histological examinations were carried out under the light or electron microscope (Sopelem, type OPL 75). In the first case the samples were fixed in form01 or in Bouin’s solution and were stained with hematoxylin and eosin, Masson’s trichrome or luxol fast blue-cresyl violet and bodian luxol fast blue. In the second one they were fixed in glutaraldehyde, postfixed with osmic acid, and contrasted with lead citrate and many1 acetate. The histological examination of thoracoabdominal organs (heart, lungs, pancreas, spleen, kidneys, ovaries, testis) and of the nervous system were carried out. The subacute toxicity was determined by the ip route. Treatment was given daily for 5 consecutive days to groups of ten animals (five males and five females). Doses were calculated according to an arithmetic progression of 2.5 in the range 5-15 (males) or 5-20 mg/kg (females). The animals were observed over a period of 1 mo, regularly weighed and submitted to hematological examinations. Blood samples were collected before treatment and every week over 1 mo. Some experiments were added to the abovementioned studies : mice were given daily ip doses of 9-OH-E during a certain period of time. Intermittent dosing was given by the ip or iv route to male and female mice. The irrjections (two or more) were spaced at experimentally defined time intervals. The mice were observed for 1 mo after first dosing and submitted to hematologic and histologic examinations. RESULTS Acute Toxicity Only po doses of 9-OH-E greater than 500 mg/kg caused deaths. The results of the toxicity studies performed by the ip and iv routes are presented in Table 1, The product showed a greater toxicity by the ip route. By the iv route, 9-OH-E was almost as toxic to males as to females, but by the ip route, it was more toxic to males. Our results indicate that the sublethal dose is close to the LD50. The steepness of this dose-response curve is not uncommon for antineoplastic agents. For each route of administration, the time of crisis was close to 8 days.

LDO (estimated)

90

140

170

Sexand numberof animals

CT+6 170

5 130

9 125

’ Dosesareexpressed in mg/kg. bProbitmethod,95%confidence limits.

ip

Route of administration iv

1

169 (164-174) 202 (194-210)

113 (109-117)

LDSOb

5

250

150 7

7

LDlOO (estimated)

200

Time of crisis (days)

24 hr after injection

ACUTE TOXICITY OF 9-Hynrzoxy-ELr.rPrrcrNEIN MICE”

TABLE

55

55

75

LDO (estimated)

68 (65-70) 84 (81-88)

102 (94-l 10)

LD50b

At time of crisis -

140

80

150

LDlOO (estimated)

z 3 Fs ? F z zt ij z 2 r ii2

$ s?

8 ij

488

CROS ET AL.

Three main types of signs were noted: hypothermia, weight loss, and locomotor disorders. Hypothermia appeared after iv or ip doses well below the estimated LDO. There was a dose-response effect from 5 to 50 mg/kg; the largest temperature changes were generally reached 1.5 hr after administration of the drug (-4.5”C for 50 mg/kg) and temperatures gradually returned to normal values after 24 hr. At 5 mg/kg the temperature of the treated animals did not differ significantly from that of the controls. The animals lost weight for 6-7 days, i.e., until the time of crisis. They subsequently recovered their normal weights. At the estimated ip LDO, weight loss reached 20”/, in males. I

120. 100

_

60

.-.-..

lQuk0cyt.l ne"trophils

----

lymphocyte.

i

,Q..'.

.\ .. ,_,C,_._.-.

-.-Q

i i

15w/kg

60 6 100

:

1

11 I

17 I

31 I

TIME

AFTER

Ii,

TKIEATMENT

I

I

(days)

2. Effect of a single iv dose of 9-hydroxy-ellipticine on leukocyte counts of female DBA/Z mice as a function of time. Each point represents the mean variation of initial percentage values determined in eight animals. FIG.

At doses which caused death in 24 hr (170 mg/kg in males, 200 mg/kg in females), paralysis of the hind legs appeared about 17 hr after treatment. At smaller doses (1 OO170 mg/kg) only a loss of equilibrium was noted. Hematologic changes were observed; the iv dose of 15 mg/kg only caused a transient reduction in polynuclear neutrophils (45 %) on day 3. At the iv dose of 75 mg/kg a far more marked response was obtained (Fig. 2). As early as day 3, reductions of about 10 % of red cell and of 45 % of leukocytes were noted. Polynuclear neutrophils were particularly affected (96% reduction) and the amount of lymphocytes was reduced

TOXICITY OF %HYDROXY-ELLIPTICINE

489

IN MICE

by 32 “/:. On day 6, the amount of lymphocytes was still low while; by contrast, the count of polynuclear neutrophils was higher than the initial value, thus revealing a phenomenon of “rebound” which suggestedthat a stimulation of somestemcells occurred. A parallel phenomenon also was noted in bone marrow smearsfrom mice given 40 and 60 mg/kg (ip); on day 2, the marrow was very poor in leukocytes and almost totally populated with erythrocytic elements, whereas as early as day 4, the marrow was already active and rich in cells with mitotic figures. The following experiments were aimed at determining: (a) at what time maximum variations appeared in leucocytes after a single injection of 9-OH-E, (b) the threshold doseat which such variations began to appear. * 40,

leukocytes -. - . - ncutrophits - - - - lymphocytes

a $

ul g a E

‘\ ‘\

60,

60

‘la .-.-.-.

+&,\*

40

50

4.

_

s t

5 ’

10

15

-*-e 25

DOSES

60

(mglkg)

FIG. 3. Effect of a single ip injection of 9-hydroxy-ellipticine on leukocyte counts of DBA/? female mice as a function of dose. Blood examinations were performed 2 days after administration of the drug. Each point represents the mean variation of initial percentage values determined in six mice for total leukocytes and in three mice for neutrophils and lymphocytes.

In the first experiment, the mice (five animals in each series)received a single ip dose of 9-OH-E (55 mg/kg). Blood sampleswere collected daily for 8 days. The number of leukocytes was considerably reduced on day 2 and then returned to its normal value after 8 days. Slightly greater doses(60 and 65 mg/kg), administered under the same conditions, confirmed these results. In the second experiment, increasing amounts of 9-OH-E (5-60 mg/kg) were given to six mice per group, and the animals were killed after 2 days. Figure 3 presents the results obtained for total leukocytes, polynuclear neutrophils, and lymphocytes. It appears that the hematologic threshold dose under which no hematologic change is recorded, is situated between 15 and 25 mg/kg, since a significant reduction in the

490

CROS

ET AL.

amount of lymphocytes occurs within this dose range. These values are three to four times higher than the therapeutic dose in L1210 tumor-bearing mice. The amounts of red cells and platelets did not vary. No histological changes were observed in the thoracoabdominal organs after sublethal doses of 9-OH-E (ip:2.5, 5, 10, 20, and 30 mg/kg; iv: 15 mg/kg). On the other hand, after a single lethal dose, the liver displayed cholestatic lesions with biliary thrombi. Evidence of damage to the nervous system was carefully looked for in mice given different dosages. Lethal doses (170 or 185 mg/kg ip) led in four mice to intense neurologic, mainly locomotor disorders followed by paraplegia, as previously described. The nervous tissues of these animals revealed diffuse neuronal alterations, varying in intensity according to their location. The cerebellum, and more especially Purkinje cells, were particularly affected, but lesions of this type were also present in the brainstem and, to a lesser extent, in the cortex and the spinal cord. These changes explain the previously described signs. Equilibrium disorders are due to neuronal alterations of the cerebellum, while locomotor disorders may be caused by neuronal lesions affecting the anterior horns of the marrow. Nissl bodies and the nuclei were likewise affected; the cytoplasm was homogenous and acidophilic; the nucleus lost its nucleolus, then became acidophilic too. The same observations apply to Purkinje cells, which became pycnotic, then could disappear, leaving empty areas sometimes filled with Bergman’s glia (Fig. 4). However, no demyelinization was observed. Sublethal doses (55 mg/kg ip, 75 mg/kg iv) led, in a minority of mice, to histologic alterations more particularly affecting the cerebellum, without any apparent functional disorder. The evolution of these changes was followed in two mice for each dose, on days 2,6 and 3 1, of treatment. The lesions seemed to stabilize as early as 48 hr. On days 6-31 only a discrete loss of neurons still persisted, with the presence of Bergman’s glia. Furthermore, for a single injection of such a moderate dose, the damage was focal and did not affect a great number of Purkinje cells. Under the electron microscope, dilations of the endoplasmic reticulum and of Golgi apparatus associated with a reduction in the number of ribosomes and an alteration of the nuclei, which were pycnotic, irregular, with deep indentations and a dilation of the perinuclear cisternae, were observed. A histological threshold dose was determined in seven series of three female mice, each series being treated with single increasing ip doses of 9-OH-E (from 5 to 60 mg/kg) and killed 4 and 8 days after treatment. The determination of the threshold dose only on the basis of histologic criteria was not accurate. However, this dose amounted to about 50 mg/kg. Subacute Toxicity

Lethal doses for a daily ip treatment given for 5 consecutive days, are indicated in Table 2. Sex differences in sensitivity appeared here too. The data revealed that, for a given amount of g-OH-E, a single dose or several fractionated doses administered at 24-hr intervals gave about the same mortality. It was thus established that 9-OH-E is a cumulative toxic substance. The experiment was carried out only by the ip route, since the product induced necrosis on the vessels when given repeatedly through the iv route. This local toxicity was not related to the pH (5) of the solution injected.

TOXICITY

OF %HYDROXY-ELLIPTICINE

FIGURE

4.

IN

MICE

49

492

CROS ET

AL.

Lethal doses of 9-OH-E mainly induced locomotor disorders which appeared in mice, as mentioned above, during the preagony phase. They were characterized by a loss of equilibrium and a paralysis of the hind legs. TABLE 2 SUBACUTE

TOXICITY

OF %HyDROxY-ELLIPTIcmE

IN MICE”

Lethality at time of crisis Sex and number of animals

Time of

6 64

15

0 80

8

crisis

(days)

LDO (estimated)

LDSO*

7.5

LDlOO (estimated)

15 &6) 20

10.0

(l&

a ip doses repeated for 5 consecutive days; doses are expressed in mg/kg. * Probit method, 95 % confidence limits. To study hematologic variations, treated animals were given daily doses of 10 and 15 mg/kg for 5 days. By the end of the first week, a IO-20 % reduction in the number of erythrocytes per mm3 was noted. This remained constant until the third week and was later restored to its normal value. The leukocyte count dropped by the end of the first week. The reduction amounted to 40-50x and polynuclear neutrophils were mainly involved (70 ‘A of reduction); they began to increase again as early as the second week. The lymphocytes were less affected (30 % of reduction), but for a longer period, since the reduction of their blood concentration still persisted after the fourth week. In the animals given sublethal doses of 9-OH-E (15 mg/kg/day ip for 4 days), no locomotor disturbance could be observed by the rotating rod test, the fixed rod test or the loss of the righting reflex. Ten or 3 1 days after the administration of 5 mg/kg for 10 days, neuronal lesions were inconstantly observed. These results suggest that some mice could be more susceptible to repeated nonlethal doses than others. It has been previously demonstrated (Le Pecq et al., 1973) that 9-OH-E is endowed with a marked antitumor activity against L1210 leukemia (single ip dose of 5 mg/kg). To provide possibletherapeutic developments to this finding, it was essentialto know how many times and at what frequency such a dose could be given without causing a

single death. Daily doses of 5 mg/kg were administered

until death of one animal per

FIG. 4. Cytology of cerebellums of 9-OH-E-treatedmice. Upperplate: cerebellumfrom normal femalemouse;molecularlayer (M), Purkinjecells(P),granularlayer(G) (x750).In inset,two normal Purkinjecells(x1700).Middle plate: cerebellum from a femalemousetreatedwith 9-OH-E (single iv injection of 75mg/kg)andkilled2 daysafter treatment.NormalandpycnoticPurkinjecellsareclose to eachother. Smallareasare partly filled with cytoplasmicfragments,which corresponds to the beginningof a neuronaldepopulation(x750). In inset,normaland pycnoticcells(x1700).Lower plates:cerebellum from a malemousetreatedwith 9-OH-E(singleip injectionof 185mg/kg)andkilled 17hr after treatment.No significantalterationof themolecularlayeror of the granularlayer.Necrosis of Purkinjecellsandproliferationof Bergman’s glia(arrow)(x750).Stainingwith Masson’s trichrome.

TOXlClTY

OF %HYDROXY-ELLIPTICINE

493

IN MICE

experimental series. The incidence of mortality was followed after the end of the treatment. The animals were observed over a period of 1 mo. This experiment showed that 11 or 12 injections were end points. Under the sameconditions, it was impossible to deliver more than nine daily dosesof 10 mg/kg. Mortality increased for 4 days and reached the values recorded in Table 3. The cumulative effect of the drug was again apparent. TABLE 3 SUBACUTE TOXICITY OF 5%HYDROXY-ELLIPTKINE Number of animals 7

Sex

Number of injections

c-7

11

6

,:;

6

L!

12 9 9

8

IN MICE" -

Daily dose (m/kg)

Total dose (mg/kg)

5 5 10 IO

55 60 90 90

Mortality” (“/O 87.5 12.5 100 16.7

’ ip doses repeated until first death. ’ Final mortality determined 4 days after end of treatment.

The product mainly induced locomotor disorders which appeared in mice during the preagony phase. The number of red cells was almost unchanged, even a short time before death. On the contrary, a severe leukopenia was noted, which could yield values as low as 1000leukocytes per mm3.

NUMBER

OF

INJECTIONS

FIG. 5. Effect of repeated ip doses of 9-hydroxy-ellipticine (5 mg/kg every 3 days) on leucocyte counts of DBA/2 female mice as a function of time. Each point represents the mean variation of initial percent values determined in five animals after a different number of injections.

494

CROS ET AL.

Six mice observed 24 hr after treatment analogous to those previously described.

presented neurologic

signs and lesions

Intermittent Dosing The interval of time between two ip doses of 9-OH-E that are close to the estimated LDO and do not cause a single death was determined. By the ip route (45 mg/kg/ injection) it was 3 days for the females and 5 days for the males; by the iv route (65 mg/kg/injection), it was 5 days for each sex. In an attempt to extend the former results to longer periods, another experiment was undertaken; ip doses of 5 mg/kg were given to female mice, every 3 days, over a period of 132 days (total dose = 220 mg/kg). During the whole experiment, no mortality was noted. It was observed that animals submitted to a long- lasting treatment of 9-OH-E often suffered from peritonitis. Repeated injections of 9-OH-E could cause local ulcerations or necrosis of tissues with secondary inflammatory phenomena. Such peritonitis was not found in the animals treated under the same conditions with a buffer, pH 5. The hematologic effects of this intermittent treatment were recorded. The results (Fig. 5) showed that only the polynuclear neutrophil counts decreased transitorily by the sixth week of treatment. Surprisingly, after the third month, the mice had normal blood concentrations of neutrophils, in spite of the continuous treatment. These results, checked by the paired t test, were found to be significant at the 5 % level. No histologic alteration was noted in groups of three mice killed after 5, 10, 15, 19, 30, and 44 injections concomitantly with nine control mice receiving only acetate buffer.

DISCUSSION

The introduction of an OH group on the ellipticine ring very markedly enhances the anti-tumor properties of this molecule. Intraperitoneal doses of 170 mg/kg of ellipticine administered to L1210 leukemic mice gave an increasing life-span (ILS) of 42 % (Venditti and Abbott, 1967), while ip doses of 50 mg/kg of 9-OH-E resulted in an ILS of 51%. Furthermore, from a toxicological point of view, hydroxylation of the molecule has a highly favorable action, for the cardiovascular and respiratory depression shown in ellipticine-treated dogs (Herman et al., 1971) is changed into a remarkable progressive and durable cardio-stimulating action in g-OH-E-treated dogs (Chanh et al., 1974). Such data led to the present work. 9-OH-E shares with three other anticancer drugs (daunomycin, adriamycin, and actinomycin D) the ability to intercalate into DNA base pairs (Festy et al., 1971). The substances also display some common toxicological features : they are not lethal by the po route, unless very high doses are used, and more toxic by the ip than by the iv route. They are endowed with marked cytotoxic properties and are potent teratogens (Dubost et al., 1964; Philips et al., 1960; Tuchmann-Duplessis and Mercier-Parot, 1960; unpublished data). However, any correlation between these common characteristics would be unwarranted and preliminary data (Le Pecq et al,, 1974b) indicate that there is no obvious relationship between the lethality and the in vitro affinity for DNA of various derivatives in the ellipticine series.

TOXICITY

OF %HYDROXY-ELLIPTICINE

IN MICE

495

Its lethality and cytoneurological effects show that 9-OH-E is a strictly cumulative toxic. It has been reported that this is a characteristic of most antitumor agents (Griswold et al., 1963; Freireich et al., 1966). The effects of a first ip injection are gone after 3-5 days, depending on sex. This must be taken into account if repeated doses were administered to humans. The dose tolerable in one intake ought to be separated into fractions given at intervals of several days, the total amount of drug remaining unchanged. Lethal doses are not very far from the maximum tolerable dose. Therefore, any dose schedule in man should be increased only with great caution. The three main signs, in mice as well as in dogs (Chanh et al., 1974), were a fall in temperature, hematological, and neurological changes. Whatever the route of administration, there was a marked fall in temperature. It may be due either to an alteration of thermoregulatory nervous centers, as suggested by neurological disturbances, or to modifications of the mitochondrion. Although no change in the morphology of mitochondria was detected in the present work, such changes have already been reported in HeLa cells exposed to 9-OH-E (Oustrin and Pieraggi, 1974). Moreover, preliminary results obtained in this laboratory, suggest that the oxidative phosphorylation of rat liver mitochondria is modified by 9-OH-E in vitro. It has been recently reported that reserpine-induced hypothermia could explain the efficiency of this alkaloid on L1210 leukemia (Fondy et al., 1974). This hypothesis is not tenable in the case of 9-OH-E because a single ip dose of 5 mgikg active against this leukemia has no significant effect on body temperature. Hematological disorders are very common to anticancer drugs. Leukocytes are particularly affected and the number of polynuclear neutrophils is markedly but transitorily reduced. These results have been confirmed in another animal species, the Wistar AG rat. Lymphopenia, which may be long-lasting, could be related to immunosuppressive effects and to the antiinflammatory action of 9-OH-E. Leukopenia occurred after different time and dose schedules. Neutrophils were especially susceptible to the drug. Injections of nonlethal doses repeated every 3 days led to an important reduction in the number of polynuclear neutrophils within 6 wk of treatment. However, very surprisingly, a prolonged treatment resulted in a reversal of this picture. After the third month, the concentration of polynuclear neutrophils was above normal values and reached control values 1 mo later. It is difficult to interpret such a recovery occurring during a continuous treatment. One interpretation might be that on chronic administration, induction of microsomal metabolism occurs. Such a phenomenon has been observed in several instances (Conney, 1967). The neurological signs resemble those of cerebellar ataxis and are very probably caused by preferential alterations of Purkinje cells (detected at histological examination). It has been already demonstrated by Liss et al. (1972) that ellipticine, which is devoid of a hydroxy group in position 9, crosses the blood-brain barrier to reach Purkinje and granular cells in the cerebellum and specifically binds to them. Four conclusions can be drawn from this neurotoxicologic study : (a) no neurological signs and cytological damage of the nervous tissues were recorded after administration of a dose of 9-OH-E active against L1210 leukemia. The threshold dose for such cytological alterations is approximately 50 mg/kg, i.e., ten times the therapeutically active dose (Le Pecq et al., 1973). It is to be noted that the above experiments yielded this value when the mice received either only one injection of this dose or several daily

496

CROS ET AL.

injections of the same fractionated total dose. (b) Animals receiving the estimated LDO and thus surviving

the treatment

presented alterations

of Purkinje

cells without

any

signs. However, in each series, only a few animals were apparently affected and had only a few damaged Purkinje cells. (c) As expected, chemically induced neurological alterations were irreversible. (d) There is a very clear discrepancy between cytological damage to the nervous tissues and neurological signs. Only mice in the preagony

phase, having received rather considerable quantities of the drug, presented apparent equilibrium and locomotor disorders. Therefore, it is advisable not to rely only on the changes in neurological behavior to evaluate the deep toxic effects of g-OH-E. Finally, these data indicate that attempts at obtaining mice devoid of Purkinje cells by repeated injections of sublethal doses of 9-OH-E could be rewarding. At present such animals can only be provided by genetical manipulations (Sidman and Green, 1970).

They could be useful for research on the physiopathology

of cerebellar functions.

ACKNOWLEDGMENTS We thank Dr. J. B. Le Pecq, Dr. Ch. Gosse, and Prof. J. Cros for fruitful discussions. Mrs. G. Franqois and H. Vinial, F. Foulquier, and J. Bonnefoux, Miss Ch. Vila, and Mrs. Ch. Gouarderes provided skillful technical assistance. The English translation of the manuscript was carried out by Miss N. Patez. REFERENCES M. (1954). Traits’ de Cytologie Sanguine. Massonet Cie, Paris. CHANH, P. H., SOKBARA, R., DAT-XUONG, N., LE PECQ, J. B. AND PAOLETTI, BESSIS,

C. (1974).Actions cardiovasculaireset toxicit& de I’hydroxy-9-ellipticine chez le chien. C. R. Acad. Sci.

(Skrie D) 279,1039-1041. CONNEY,

A. H. (1967). Pharmacologicalimplications of microsomal enzyme induction.

Pharmacol.

Rev. 19, 317-366.

K., DEMERAC, S., ELMES, B. C., LODER, J. W., SWAN, J. M. AND TEITEI, T. (1967). Synthesisof the tumor-inhibitory alkaloids, ellipticine, 9-methoxyellipticine, and related pyrido[4-3,blcarbazoles.Aust. J. Chem. 20, 271 S-2727. DUBOST, M., GANTER, P., MARAL, R., NINET, L., PINNERT, S.,PREUD’HOMME, J. AND WERNER, G. H. (1964). Rubidomycin: a new antibiotic with cytostatic properties. Cancer ChemoDALTON,

L.

therap. Rep. 41, 35-36. FESTY,

B., POISSON,

J. AND

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Toxicological and pharmacological studies on 9-hydroxy-ellipticine in mice.

TOXICOLOGY AND APPLIEDPHARMACOLOGY33,484-497(1975) Toxicological and Pharmacological Studies 9-Hydroxy-Ellipticine in Mice on S. CROS,~ R. SORBARA...
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