J Cancer Res Clin Oncol (1996) 122:102-108 �9 Springer-Verlag 1996

R. A. Schwendener �9 D. H. Horber �9 B. Odermatt H. Schott

Oral antitumour activity in murine L1210 leukaemia and pharmacological properties of liposome formulations of N 4-alkyl derivatives of l-p-D-arabinofuranosylcytosine

Received: 23 May 1995/Accepted: 18 July 1995

Abstract The oral cytostatic activity in L1210 mouse leukaemia of the two new N4-alkyl derivatives of 1-fi- D-arabinofuranosylcytosine (AraC), Nr - and N4-octadecyl - 1-fi-D-arabinofuranosylcytosine (NH- AraC, NO-AraC) was investigated. In contrast to AraC, both derivatives were highly cytostatic after oral application as liposome formulations. With treatment schedules of five consecutive dosages or with two ap- plications on days 1 and 4 after intravenous tumour cell inoculation with a total dose of 470-1000 mg/kg NH- AraC or NO-AraC, 70% 100% of the treated animals were cured. The lethal dose in healthy ICR mice after a single intraperitoneal application, corresponding to the LDs0, was 524 mg/kg for NO-AraC, whereas NH- AraC proved to be less toxic. The haematological toxi- city remained moderate for both drugs with a mild leucopenia and a drop in platelet counts, which re- covered 4-6 days after treatment. The erythrocytes were not affected and haemolytic toxicities were absent. As non-haematological toxicities, at high drug concen- trations, a pronounced atrophy of the rapidly dividing epithelia of the small intestines and of the white pulp of the spleen were observed. The blood levels of NH-AraC given orally reached values comparable to those after parenteral application of a four-times lower dose of NH-AraC, suggesting a moderate bioavailability. Thus, these two lipophilic derivatives of AraC are compounds

R. A. Schwendener (5:~) . D. H. Horber Department of Internal Medicine, Division of Oncology, University Hospital, R~imistrasse 100, CH-8091 Zfirich, Switzerland. Fax: 0041 1 255 44 20

B. Odermatt Department of Pathology, University Hospital, CH-8091 Ztirich, Switzerland

H. Schott Institute of Organic Chemistry, University of Ttibingen, D-72076 Tiibingen, Germany

with a potential for the oral treatment of malignant diseases.

Key words Lipophilic AraC derivatives �9 Oral activity �9 Toxici ty. Pharmacokinetics �9 Liposomes

Abbreviations NH-AraC N4-hexadecy1-1-/~-D-arabino- furanosylcytosine - NO-AraC N*-octadecyl-1-/~-D - arabinofuranosylcytosine �9 AraC 1-/~-D-arabinofuran- osylcytosine

Introduction

The nucleoside analogue 1-fl-D-arabinofuranosyl- cytosine (AraC) is one of the most important cytostatic agents for the treatment of leukaemias and lymphomas (Chabner 1990; Keating et al. 1992). In addition, AraC has been evaluated for the treatment of myelodysplastic syndrome as a low-dose therapy regimen (Hellstr6m- Lindberg et al. 1994; Miller et al. 1992). One of the major clinical disadvantages of AraC is its ineffective- ness after oral application (Neil et al. 1970; Ho and Frei 1971), which is caused by poor absorption, fast elimina- tion and, what is more important, by fast deamination by pyrimidine aminohydrolase to the inactive metab- olite 1-fi-D-arabinofuranosyluracil (AraU; Ho et al. 1975).

To increase the stability of AraC various attempts have been made to alter the nucleoside chemically into more stable derivatives that would also permit the application of the drug by the oral route. A great number of N 4- and Y-derivatives of AraC have been synthesized and their suitability to replace AraC was investigated (Wempen et al. 1968; Tsuruo et al. 1980; Rosowsky et al. 1982). However, none of these deriva- tives was able to substitute AraC in the clinically applied parenteral treatment modalities. More recently, new strategies have been followed and the potential usefulness of lipophilic derivatives of AraC as orally

active drugs has been described. Ohno et al. (1986) reported a phase I study with the derivative Nr - mitoyl-AraC and Kodama et al. (1989) and Ohno et al. (1991) introduced 1-fi-D-arabinofuranosylcytosine-5'- stearyl phosphate (fosteabine) as an orally active deriv- ative of AraC.

In earlier studies, we demonstrated the excellent anti- tumour effects of liposome preparations of N4-acyl - AraC derivatives, which had a higher activity than the parent drug (Rubas et al. 1986). With the synthesis of N4-alkyl-AraC derivatives, we obtained compounds of extremely high stability against deamination and of further increased anti tumour activity (Schwendener and Schott 1992; Schott et al. 1994; Schwendener et al. 1995). Pharmacological studies with liposome formula- tions of the N~-hexadecyl-l-fi-D-arabinofuranosyl - cytosine derivative (NH-AraC) revealed that this new compound exerts its cytotoxic action by mechanisms that are, to a large extent, different from those of AraC (Horber et al. 1995a-c).

In this work we report the oral anti tumour effects of the Nr derivatives, NH-AraC and N4-octa - decyl- 1 -fi-D-arabinofuranosylcytosine (NO-AraC), and describe their acute toxicities, haemolytic activities, and the pharmacokinetic parameters of NH-AraC.

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extruded through 0.1 lam membranes. The NH-AraC-liposomes were trace-labelled with [5-3H] NH-AraC for detection and quan- tification. As a comparison to the liposome preparations, NH-AraC was also suspended in peanut oil at 40 mg/ml for oral application in the L1210 leukaemia model. AraC was freshly dissolved in phos- phate buffer at 40 mg/ml.

Oral antitumour activity against L1210 leukaemia

The antitumour activity of the liposomal drug preparations was evaluated with the murine L1210 leukaemia model in BDF1 mice (Schwendener and Schott 1992). The L1210 cells (1 x l0 s in 0.1 ml) from donor ascites were injected intravenously on day 0. Groups of 5 7 BDF1 mice (Charles River, Sulzfeld, Germany; average body weight 20.6 + 0.6 g) were kept in Makrolon type-3 cages and fed with water and solid diet at libitum. Before each treatment the mice were fasted overnight. Drug treatment was performed by the use of a stomach catheter. A volume of 0.5 ml liposomes containing NH- AraC or NO-AraC (extruded through 0.4 btm) was applied accord- ing to the following schedules: (a) five consecutive applications on days 1-5 or, (b) two treatments on days 1 and 4 after tumour cell injection. In one experiment NH-AraC was also given orally as a crystalline suspension in peanut oil. Control groups received AraC in 0.5 ml phosphate buffer or were left untreated. Increase of lifespan of treated animals (T) was evaluated by recording the mean survival time as compared to the untreated controls (C), expressed as T/C as a percentage (Table 1). The animals were observed daily until death or for 60 days. Animals surviving 60 days were included into the calculation and considered as being cured.

Materials and methods

Lipids and chemicals

Soy phosphatidylcholine was obtained from L. Meyer, Hamburg, Germany. Cholesterol, recrystallized from methanol, was from Fluka AG, Buchs, Switzerland. DL-e-Tocopherol and all analytical- grade buffer salts were from Merck, Darmstadt, Germany. N 4- Hexadecyl- and N4-octadecyl - 1-/?-D-arabinofuranosylcytosine (NH- AraC, NO-AraC) were synthesized as described before (Schwen- dener and Schott 1992; Schott et al. 1994). Tritium-labelled N4-hexadecyl- 1-fi-D-arabinofuranosylcytosine (5-[3H]NH-AraC, 0.189GBq/mmol) was from Amersham Int., Amersham, UK. Tritium-labelled NO-AraC was not available. All other chemicals were of analytical grade (Merck).

Liposome preparation

Unilamellar liposomes were prepared by filter extrusion of multi- lamellar liposome suspensions using a Lipex extruder (Lipex Biomembranes Inc., Vancouver, Canada) as described by Hope et al. (1985). They were composed of soy phosphaditylcholine (40 mg/ml)/cholesterol (4 mg/ml)/DL-~-Tocopherol (0.2 mg/ml)/ at a molar ratio of 1:0.2:0.01. For the oral treatment experiments the lipophilic derivatives NH-AraC or NO-AraC were added at 8 mg/ml to the lipids in the organic phase (methanol/methylene chloride, 1 : 1, v/v). After evaporation of the organic solvents at 40~ multilamellar liposomes were obtained by addition of phosphate buffer (67 raM, pH 7.4). After extrusion through Nuclepore membranes (Costar, Sterico, Dietikon, Switzerland) of 0.4 btm the liposomes were concen- trated in an Amicon ultrafiltration cell (Amicon Corp., Lexington, Mass.; Diaflo YM membranes, Mr-100000 cut-off) to obtain drug concentrations of 30-40 mg/ml for the oral treatment experiments. The preparations used for the toxicity experiments were additionally

Acute toxicity of NH-AraC and NO-AraC after intraperitoneal treatment

The acute toxicity of NH-AraC and NO-AraC was determined according to OECD guideline 420 in healthy mice (van den Heuvel et al. 1990). Liposome preparations of NH-AraC or NO-AraC (extruded through 0.1 btm) were injected intraperitoneally (i.p.) as single dose in 1 2 ml volume to groups of 3 6 ICR mice (20 g average weight, females or males). The intraperitoneal application route had to be chosen because of the following constraints faced with intravenous and/or oral drug application. Intravenous drug injection in mice is limited by the injection volume which is maxi- mally 0.2 ml. Correspondingly, it was not possible to treat mice orally with volumes exceeding 0.5 ml with high drug concentrations (e.g. 10 mg NOAC corresponding to 524 mg/kg). Therefore, for the estimation of the toxic dose of NO-AraC a pilot experiment was performed in which mice were given doses of 250,500, and 750 mg/kg NO-AraC in a volume of 1-2 ml by i.p. application. According to the outcome of the test experiment, the acute toxicity of the two derivatives was determined by treating mice i.p. (six per group, females and males) with doses of 524 mg NH-AraC or 524 mg and 380 mg NO-AraC/kg body weight (Fig. 2a,b). The weight of the mice was recorded before treatment and daily thereafter and com- pared to untreated control groups (three per group). The mice were observed daily and changes in behaviour and other symptoms were recorded.

Haematological toxicity of NH-AraC and NO-AraC

The haematological toxicity of NH-AraC and NO-AraC was deter- mined in healthy female ICR mice after a single i.p. treatment with the sublethal doses of 400 mg/kg NH-AraC and 350 mg/kg NO- AraC, respectively. To groups of three mice the liposomal drugs were given i.p. on days - 10, - 8, - 6, - 4, - 2, and 0. All mice were killed on day 0 and blood was collected into heparinized tubes

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(Vacutainer, Becton-Dickinson) by heart puncture. The haema- tological parameters such as white blood cell count, red blood cell count, and platelet count were measured with a Contraves Autolyzer 820 (Contraves AG, Zfirich).

Haemolytic activity of NH-AraC and NO-AraC in vitro

Liposome preparations of NH-AraC and NO-AraC were incubated at different concentrations (0.2-4 raM) with freshly collected human blood from healthy donors for 60 min at 37~ Aliquots of the supernatants obtained after centrifugation (15009, 30min) were diluted 1:100 in 0.9% NaCI and the concentration of haemoglobin was determined by calculating the difference between the absorb- ance at 577 nm and 561 am. Total haemolysis (100%) was obtained by incubation of erythrocytes in water containing 0.02% Triton X-100 (Sigma, St. Louis, Mo.) at a 1:1 (v/v) ratio.

Histological examination of NO-AraC toxicity

The organ toxicity in mice treated i.p. with a dose of 524 mg/kg NO-AraC was documented by removing liver, spleen, kidneys and small intestines shortly before they had to be killed because of lethal drug toxicity (cf. Fig. 2a, b). Occasionally histology was also per- formed with mice treated with sublethal drug dosages. To obtain histological sections the organs were rinsed in phosphate buffer, fixed in the same buffer containing 4% formaldehyde and embedded in paraffin according to standard procedures. Serial sections of 4 pm thickness were mounted on glass slides and counterstained with haematoxylin and eosin. The sections were examined with an Olym- pus microscope under bright field.

Pharmacokinetic properties of NH-AraC

Liposomal NH-AraC (12 rag, corresponding to 25 bLmol/kg NH- AraC body weight) and trace-labelled with [5-3H] NH-AraC (12 kBq/animal) was given orally in a volume of 0.5 ml to BDF1 mice (20-25 g, females). After periods ranging from 1 h to 24 h, groups of three mice were killed and blood, liver, spleen, kidneys and small intestines were removed. Whole, weighed organs were digested

with 2-4 ml Soluene 350 (Packard) tissue solubilizer at 40 50~ until total solubilization of the organs. The blood samples were solubilized with 2ml 1:1 (v/v) mixture of Soluene 350 and iso- propanol. The solubilized samples were bleached by dropwise addi- tion of 0.5-1 ml hydrogen peroxide 30% under cooling in an ice bath, neutralized with 0.1 ml 10% HC1 solution and counted after addition of 10-15 ml Hionic-Fluor (Packard) scintillation cocktail. The total blood volume was calculated as 7.7% of the body weight and the concentrations of NH-AraC in liver, spleen and kidneys were corrected for blood contamination according to Allen (1989).

Results

Antitumour activity after oral application

The antitumour effects against L1210 leukaemia of NH-AraC and NO-AraC after oral drug application are shown in Fig. 1 and summarized in Table 1. In Fig. 1 the survival curves of mice treated with liposomal NH-AraC and NO-AraC are compared with untreated controls and with animals treated with an equimolar concentration of AraC given in phosphate buffer. Both N~-alkyl derivatives were active after oral treatment, whereby five consecutive drug applications (on days 1-5) resulted in better antitumour activities as compared to the schedule where the same total dose was given on days 1 and 4. Both AraC derivatives were highly active at 1 mmol/kg or 2 mmol/kg total dose, resulting in 70%-100% cured animals. As well documented (Neil et al. 1970; Ho and Frei 1971), AraC given by the oral route was only marginally active, yielding T/C values of 160%-180% without any surviving animals. In Table 1 the cytostatic effects are summarized and the application of NH-AraC in lipo- somes is compared with a suspension of the drug in oil. The oil suspension was less active with both dosage

Table 1 Oral treatment of L1210 leukaemia with lipophilic derivatives of cytosine arabinoside (AraC) a. NH-AraC N4-hexadecyl-1 - fi-D-arabinofuranosylcytosine, N O- AraC N4-octadecyl-l-fl-D - arabinofuranosylcytosine, PB phosphate buffer (67 mM, pH 7.4)

Preparation Schedule b Total dose Survival time (days) T/C c Survivors (%) 60 days

(mmol/kg) (mg/kg) Range Mean + SD

NH-AraC in liposomes qdl 5 1 2

d l + 4 2

NH-AraC in oil a qdl 5 2 dl + 4 2

NO-AraC in liposomes @1-5 1 2

AraC in PB qdl -5 2 4

Controls

468 13- > 60 47 • 22 600 5/7 936 > 60 > 60 7 8 9 6/6

936 13- > 60 33 • 22 437 2/5

936 22- > 60 45 • 21 589 3/5 936 2 2 - > 6 0 3 8 • 500 2/5

496 31- > 60 54 • 15 710 5/6 992 23- > 60 55 • 12 720 5/6

243 10- > 16 12 • 2 160 0/6 486 12- > 16 14 • 1.5 180 0/6

7 9 7 .6• 100 0/6

a On day 0, 10 s L1210 cells were injected intravenously into BDF1 b Total dose given either on 5 consecutive days (qdl-5) or on days ~ Increase of lifespan T/C(%) calculated including 60-day survivors d NH-AraC was given as crystalline suspension in peanut oil

mice 1 a n d 4 ( d l +4)

1 O0

90-"

8 0

o~ 70"

03 602 E "~ 5O 2.

40

"-~ 30 o3

20

10

0 0

- - i

I NH-AraC qdl-5, 2 mmol/kg I

NO-AraC qdl-5, 2 mmol/kg

I__.

Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NH-AraC d1+4, 2 mmol/kg

, 1

' '0 ' ' 2'0 ' ' ' 3'0 4 0 5'0 6'0

Days

Fig. 1 Survival curves of L1210 leukaemic mice (10 s L1210 cells given i.v. on day 0, five to seven mice per t rea tment group) after oral therapy either on days 1-5 (qdl-5) or on days 1 + 4 (dl + 4) with AraC and the N4-alkyl-AraC derivatives N H - A r a C and N O - A r a C in liposomes. AraC was given in phospha te buffer. Control animals were not t reated

schedules. In spite of the high probability that the NH-AraC-liposomes are converted to micelles in the small intestines by the action of the bile salts, the resorption of the drug seemed to be superior to that of the oil suspension, resulting in increased cytotoxic ac- tivity. However, compared with the dosage of lipo- some-incorporated NH-AraC or NO-AraC adminis- tered intravenously on days 2 and 6 after tumour cell injection, a 10- to 20-fold higher drug concentration was required with the oral administration to cure 80% 100% of all animals treated (Schwendener et al. 1995).

Acute toxicity

The acute toxicity of NH-AraC and NO-AraC was assessed in healthy ICR mice after a single intra- peritoneal (i.p.) drug application. According to the weight-loss curves shown for NO-AraC in Fig. 2a, b the dose of acute toxicity was determined as 524 mg/kg,

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which corresponds to 1.05 mmol/kg NO-AraC. At this concentration, 50% 60% of the treated animals died within 4 5 days, thus this dose can be considered as the acute LDso. Maximal loss of weight 5 days after drug treatment ranged between 30% and 40%. The mice for which NO-AraC was lethal died from cachexia, where- as those that survived the treatment recovered well and showed no toxic signs. NH-AraC given at 524 mg/kg (1.12 mmol/kg) was not toxic. In both female and male mice the maximal weight loss ranged between 7% and 13 % and all animals (6/6) survived the treatment (data not shown). The dose of lethal toxicity of NH-AraC was not determined.

Haematological toxicity of NH-AraC and NO-AraC

The courses of white blood cell and platelet counts up to 10 days after i.p. therapy with sublethal doses of liposomal NH-AraC (400 mg/kg) or NO-AraC (350 mg/kg) are shown in Fig. 3. The white cell count dropped only moderately, reaching the nadir on day 4, whereas the effect on the platelets was more pro- nounced. Both AraC derivatives caused a platelet reduction of 50% after 6 days, followed by a rapid recovery to normal values on day 8. The number of red blood cells remained unchanged over the period of observation (data not shown).

The haemolytic effect of NH-AraC and NO-AraC was measured in vitro by incubations of the drug con- taining liposomes with fresh human blood. The haemolytic activity of NH-AraC remained below 2% in the concentration range of 0.1 4 mM whereas with NO-AraC 3%-9% of the erythrocytes were lysed above 2 raM. This weak lytic activity, which is manifest only at high drug concentrations, can be explained by the very low solubility of both derivatives in aqueous media (less than 0.05 mg/ml H20) and the absence of amphiphilic properties of both compounds. The more pronounced toxicity of NO-AraC, however (Fig. 2a, b), might be caused by its slightly higher haemolytic activity.

Fig. 2a, b Course of weight 15o. changes in ICR mice after 440- a single intraperi toneal application of l iposomai N O - 43o. AraC. a Female mice treated o~ 42o. with 524 mg/kg N O - A r a C ( I , ~ 140. n = 6, 2/6 survivors), 380mg/kg _~ ~ loo- N O - A r a C (A, n = 6, 6/6 survivors), and untreated 90.

controls (�9 n = 3). b Male mice N a0- treated with 524 mg/kg N O - 70- AraC ( I , n = 6, 3/6 survivors), 60. and untreated controls (�9 n = 3)

Time (h)

150-

140"

130-

42o- ~ 1 1 0 -

2 ioo~ o

90-

7o 6o

b

time (h)

106

Fig. 3 Course of white blood cell (WBC) and platelet counts in ICR mice after a single intraperitoneal application of liposomal NO-AraC (350 mg/kg: �9 white cell counts; [] platelet counts) and NH- AraC (400 mg/kg: �9 white cell counts; �9 platelet counts)

12-

11-

10-

9-

8-

~- 7-

0 6- X

5- 0 ,'n 4-

3-

2-

i i i i i i

1 ~ 3 ~ 5 e -~ 8 ; 1'o

Days after therapy

-1500

-1250

-1000 "D

-750

MOO ~w

~50

-0 i

11

Fig. 4a,b Histological sections of the small intestines from female ICR mice. a Untreated control; b after a single i.p. treatment with 524 mg/kg liposomal NO-AraC, corresponding to the LDso. Histological organ examination was performed with lethally ill animals (see Fig. 2)

Histological examination of NO-AraC toxicity

Histologically, the toxicity of NO-AraC given i.p. in liposomes at the LDso dosage of 524 mg/kg was mani- fested mainly in epithelial cells. Changes were most prominent in rapidly dividing epithelia and consisted of atrophy with regions of initial regeneration. As shown in Fig. 4, the slender villi of the small intestine, lined by columnar epithelial cells covered by a brush border (Fig. 4a) disappeared almost completely. The distended crypts of Lieberkiihn ended in plump protrusions covered by ballooned mucoidal cells with vacuolized cytoplasm. The nuclei of crypt cells frequently exhib- ited prominent atypia. The entire mucosa was infil-

trated by inflammatory cells (Fig. 4b). Altogether, these changes represent the classical toxic signs of a drug that inhibits cell division. Likewise, the cylindrical ciliated cells of bronchioles flattened and exhibited a smooth surface towards the lumen. Epithelial cells of proximal and distal convoluted tubules of the kidneys showed a moderate degree of atrophy. The cells of the zona fasciculata of the adrenal cortex lost cohesion and displayed a more eosiniphilic cytoplasm as a sign of lipid depletion. In the liver, the size of hepatocytes was reduced and many cells containing one or two small nuclei were observed. As judged from the reduced cell density in the spleen, lymphopoiesis and haematopoi- esis were also affected. A diffuse atrophy of the white

107

12.5-

~_E 10.0

0 7.5 ,<

5.0 "6

2.5-

0.0

I v 4 .

; . . . . . . . ; , , , ; ; . . . . . . . . . . . . . . . . . . . . . 2 1'0 '1~2 ' ' 1'4 1'6 1'8 2~0 2'2 2'4

Time (h)

Fig. g Blood levels of NH-AraC after intravenous injection of 3 mg/kg ( , ) and oral therapy with 12 mg/kg (~). Liposomal prep- arations containing 5-3H-labelled NH-AraC were used and the drug concentrations in the blood determined by scintillation counting

pulp was detected, whereas in the red pulp the most prominent feature consisted in a significant reduction in the number of megacaryocytes. The toxic symptoms in the spleen correlate with the reduction of platelets as revealed by the haematological examination (Fig. 3). Comparable, but less pronounced effects on small-in- testine toxicity were found 24 h after the oral applica- tion of 56 mg/kg NO-AraC (data not shown). However, 48 h after treatment the damaged intestinal structures were regenerated.

Pharmacokinetic parameters and organ distribution of NH-AraC

In Fig. 5 the blood levels of NH-AraC after oral treat- ment with 12 mg (25 gmol/kg) are compared with the corresponding curve obtained after i.v. injection of 3 mg (6 gmol/kg) (Horber et al. 1995a). Following oral application, the blood levels reached slightly higher values, such as those after the i.v. application of a four times lower concentration of NH-AraC. These blood levels of NH-AraC, which persisted over a long period of time, might account for the antitumour effect after oral therapy. The measurement of the organ distribu- tion of NH-AraC after oral treatment revealed that a major part of the drug remained in the small intes- tines. After 1 h 20 __ 10% of the given dose was found in the small intestines and the level remained between 8% and 2% throughout the period of observation. The fraction of NH-AraC determined in the blood did not exceed 0.5% of the dose applied and the levels in liver (1%-2%), spleen (less than 0.05%) and kidneys (less than 0.5 %) remained comparably low. These low drug concentrations measured in the major organs indicate that the bioavailability of NH-AraC remains low, prob- ably at less than 20%. However, at higher dosages

(1-2 mmol/kg), the amount of orally absorbed NH- AraC was high enough to exert the pronounced anti- tumour effects as shown in Fig. 1 and Table 1.

Discussion

Our results demonstrate that, with the modification of the N4-amino group of AraC with long C16 and C18 alkyl chains, derivatives of excellent oral anti- tumour activity are obtained. In contrast to AraC, which is susceptible to enzymatic deamination, the lipophilic derivatives NH-AraC and NO-AraC are highly resistant against deamination (Horber et al. 1995b; Schwendener et al. 1995). Together with the lipophilic properties of both derivatives, their stability against deamination might contribute to the oral anti- tumour activity. In contrast to the Y-modified AraC derivative 1-fi-D-arabinofuranosylcytosine-5'-stearyl phosphate, the NCamino group of which is not pro- tected and which is metabolized to AraC and AraU (Ueda et al. 1994), our AraC derivatives were found to exert their cytostatic effects by new mechanisms of action. We found that only a small proportion of 2% 5% of the derivatives taken up by tumour cells are converted to AraC and its phosphorylated metabolites (Horber et al. 1995b). Thus, through their own mecha- nisms of action NH-AraC and NO-AraC can not be considered as prodrugs of AraC. The nature of the drug formulation, on the other hand, also seems to influence the intestinal resorption. With liposomal drug prepara- tions of NH-AraC better antitumour effects were ob- tained as compared to an oil suspension (Table 1). Although it is known that liposomes are converted to micelles by bile salts after oral application (Thomson et al. 1993), the intestinal resorption of NH-AraC in mixed lipid/cholesterol micelles seemed to be more effective than the resorption from an oil suspension.

The acute toxicity of NH-AraC and NO-AraC in healthy mice had to be evaluated after i.p. drug admin- istration because of the volume restrictions mentioned (cf. Materials and methods). The study revealed that NO-AraC is more toxic than its C~6 analogue (Fig. 2). The dose of 524 mg/kg NO-AraC given i.p. equals 1.05 mmol/kg, which corresponds to the oral dose where five out of six L1210-1eukaemia-bearing mice were cured (Table 1). Therefore, further experiments with different doses and schedules for oral NO-AraC will be necessary for the evaluation of the therapeutic index and the bioavailability of the drug and to charac- terize organ toxicity further.

When the toxic dose of NO-AraC is compared with the therapeutically active concentration after i.v. ad- ministration, which ranges from 25 to 50mg/kg (50-100~tmol/kg) in the L1210 leukaemia model, a therapeutic index of 10-20 for NO-AraC results (Schwendener et al. 1995), attesting the safety of

108

p a r e n t e r a l t r e a t m e n t schedules . The h a e m a t o l o g i c a l ef- fects of the two de r iva t ives were mi ld , the d r o p of p la t e l e t c o u n t s 6 days af ter t h e r a p y be ing the m o s t p r o m i n e n t . In c o n t r a s t to fos teab ine , wh ich was f o u n d to cause h a e m o l y s i s (Braess et al. 1994, a n d o u r o w n obse rva t ions ) , the N 4 - a l k y l - A r a C de r iva t ives a re n o t h a e m o l y t i c - a p r o p e r t y tha t inc reases the i r safety con- s iderab ly . The su i t ab i l i t y of o ra l a p p l i c a t i o n s of N O - A r a C , however , can on ly be a s c e r t a i n e d conc lus ive ly wi th fu r the r i nves t i ga t i ons on the d r u g ' s p r o n o u n c e d tox ic i ty on ep i the l i a l cells (Fig. 4). A d d i t i o n a l s tudies on the p h a r m a c o k i n e t i c s , m e t a b o l i s m , a n d ce l lu la r p h a r - m a c o l o g y of these i n t e r e s t i ng new c o m p o u n d s are in p r o g r e s s a n d will be r e p o r t e d elsewhere.

Acknowledgements The authors thank R. Zahner and H. Lutz for their assistance. This work was supported by the Sassella Founda- tion, Stiftung fiir experimentelle Krebsforschung, Stiftung zur KrebsbekS_mpfung and the Swiss National Science Foundation (grant 32-29979.90)

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