Journal of Experimental Therapeutics and Oncology, Vol. 5, pp. 15-22 Reprints available directly from the publisher Photocopying permitted by license only
© 2005 Old City Publishing, Inc. Published by license under the OCP Science imprint, a member of the Old City Publishing Group.
Antitumor activity of Nitronaphthal-NU, a novel mixed – function agent* Suva Samanta1, Anindita Pain1, Sushanta Dutta1, Ajit Kumar Saxena2, Mutiah Shanmugavel2, Renu Moti Pandita2, Gulam Nabi Qazi2 and Utpal Sanyal1 1
Department of Anticancer Drug Development, Chittaranjan National Cancer Institute, Calcutta -700026, India
2
Pharmacology Division, Regional Research Laboratory, Canal Road, Jammu-Tawi - 180001, India
Correspondence to: Dr. Utpal Sanyal, Department of Anticancer Drug Development, Chittaranjan National Cancer Institute, Calcutta – 700026, India. E-mail:
[email protected] (Received November 16, 2004; accepted March11, 2005)
Nitronaphthal-NU (Compound 1) was synthesized as a mixed-function antitumor agent based on the structures of the clinical drug CCNU and experimental compound Mitonafide. In vitro screening in four human tumor cell lines namely SNB-78 CNS, HOP-62 Lung, T47D Breast and SiHa – cervix revealed significant cytotoxicity in the former two cell lines much greater than CCNU and comparable to Mitonafide used as standards. In vivo antitumoral potency assessed in the murine ascites tumors Sarcoma-180 (S-180) and Ehrlich ascites carcinoma (EAC) by measuring the increase in median survival times of drug treated (T) over untreated control (C) mice, revealed highly significant (p<0.001) tumor regression effects greater than standards. Life span of mice bearing advanced tumor for 10 days before the drug challenge was also considerably increased. Its toxicity was assessed in vivo in normal and S-180 bearing mice by measuring drug-induced changes in haematological parameters, femoral bone marrow and splenic cellularities as well as hepatotoxicity and nephrotoxicity sequentially on days 9, 15 and 21 following drug treatment at the optimum dose of 50 mg/kg from day 1 to 7. Results indicate that it did not adversely affect haematopoiesis. The other parameters were within normal limit. The compound comparable to standards inhibited the synthesis of DNA and RNA in S-180 tumor cells.
Key words: new nitrosourea; antitumor agent; tumor cells; cytotoxicity assay; in vivo assay
INTRODUCTION
R
NHCON
Cl
NO R = H,
CCNU
= Me, Me-CCNU Structure A 2-Chloroethylnitrosoureas (NUs) like CCNU, MeCCNU (Structure A), BCNU, chlorozotocin etc find wide application (1) in the management of various human cancers. Efforts are ongoing globally to develop new nitrosoureas (2,3) that may exhibit better therapeutic efficacy having lower toxicity. There are reports that some substituted naphthalimides containing N–(2,2-
*The subjects of the above findings are covered under pending Indian Patent Application. Journal of Experimental Therapeutics and Oncology
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Samanta et al.
O
O N
N
N
NHCON NO
O
O
NO2
NO2
dimethylaminoethyl) chain best represented by Amonafide and Mitonafide (Structure B) bind with DNA by intercalation and have exhibited substantial anticancer activities in various animal tumors (4). Those have also undergone clinical trials (5,6). Hence we have chosen the naphthalimide ring residue as present in Mitonafide as the carrier molecule for the NU group. The synthesis, antitumoral and toxicological evaluations of the new nitrosourea compound Nitronaphthal-NU (Compound 1) were described here. It is hypothesised that upon in vivo enzymatic degradation, 1 may exert synergistic activity since the naphthalimide ring may bind to DNA while the NU group will exert cytotoxicity through alkylation of biological nucleophiles.
MATERIALS AND METHODS Chemicals 5-Nitro-2-[2-{3-(2-Chloroethyl)-3nitrosoureido}ethyl]-1H-benz[de]isoquinoline-1,3dione (1): To a clear solution of the 5-nitro-2-(2-aminoethyl)naphthalimide (7) (856 mg, 3 mmol) in chloroform (7.5 ml) was added 2-chloroethyl isocyanate (0.28 ml, 3.3 mmol) at a time at room temperature. A precipitate appeared immediately. It was stirred further for 3 hr. The solid was filtered and washed with chloroform to furnish the desired ureido product as powder that was further purified by crystallisation from methanol. Yield 604 mg (51.6%), m.p. 220-2220C. PMR (d6-DMSO) δ 3.13 (m, 2H, CH2N), 3.32 (m, 4H, CH2N and CH2Cl), 4.14 [m, 2H, CH2N(CO)2], 6.07 and 6.18 (t, 2H, 2 x NH), 8.04 (m,1H, arom. H), 8.67 (d,1H, arom. H), 8.77 (d,1H, arom. H), 8.95 (s,1H, arom. H), 9.48 (s,1H, arom. H). To a cooled (00C) solution of the above compound (390 mg, 1 mmol) in formic acid (6 ml) was added sodium nitrite solution (75 mg, 1.1 mmol) in water (0.7 ml) drop wise. A solid precipitate began to appear after an hour. It was stirred further for 2 hr. The solid was filtered, washed with water and dried. This was purified
Journal of Experimental Therapeutics and Oncology
A
B Nitronaphthal-NU, Compound 1
Structure B Mitonafide
16
Cl
Vol. 5
by column chromatography over silica gel (7 g). Elution with chloroform followed by repeated crystallisation from chloroform furnished desired compound 1 as a light yellow coloured solid homogeneous in TLC and HPLC. Yield 220 mg (52.5 %), m.p. 130–131ºC. HPLC: retention time 8.1 min (CH3CN:H2O, v/v 1:1). PMR (CDCl3) δ 3.53 (m, 2H, CH2N), 3.67 (m, 2H, CH2N), 4.33 [m, 4H, CH2N(CO)2 and CH2Cl], 7.23 (m,1H, NH), 7.94 (t,1H, arom. H), 8.43 (t,1H, arom. H), 8.77 (m,1H, arom. H), 9.14 (t,1H, arom. H), 9.30 (q,1H, arom. H), Mass: 420 (M+), 355 (100%), 339, 307, 269. PMR and Mass spectral analyses were carried out respectively in a Bruker 300-DPX spectrometer and in a JEOL JMS600 instrument by FAB+ method using mNBA matrix compound. Purity was checked with Waters HPLC system at ambient temperature (M-510 solvent delivery pump; 696 Photodiode Array detector set at 255 nm; µ-bondapak C18 steel column, 30 cm x 3.9 mm; isocratic mobile phase acetonitrile-water in varying proportions at a flow rate of 1.0 ml/min; data analysis in Millennium 32 Chromatography Manager). CCNU was purchased from Charles Pharma Inc., USA. In vitro screening in human tumor lines Compounds were screened by sulforhodamine-B (SRB) semi-automated assay (8) in the following human tumor cell lines SNB-78 CNS, HOP-62 Lung, T47D Breast and SiHa – cervix obtained either from National Center for Cell Science (NCCS), Pune, India or National Cancer Institute, MD, USA. Each compound was tested in triplicate sets of experiments at 10 –4 to 10 –5 M concentrations (Table 1). Stock solutions (1x10 -2 M) of test materials prepared in dimethyl sulphoxide (DMSO), were serially diluted with complete growth medium to get working solutions of 2x10 -4M, 1x10 -4M & 2x10 -5M so as to obtain final concentration of 1x10 -4M, 5x10 -5M & 1x10-5M respectively. Tumor cells were grown in tissue culture flasks in growth medium (RPMI-1640 with 2 mM glutamine,
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Nitronaphthal-NU as a novel antitumor agent
Table 1: In vitro screening data in human tumor cell lines. COMPOUND
Dose (in molar concentration) 5 x 10–5
10–5
10–4
1 CCNU Mitonafide
(1) SNB-78 CNS 65 a NA b 50
77 37 60
75 82 62
1 CCNU Mitonafide
(2) HOP-62 Lung 67 NA 70
85 40 72
88 65 73
1 CCNU Mitonafide
(3) T47D Breast 37 NA 47
52 21 51
53 41 57
1 CCNU Mitonafide
(4) SiHa Cervix 26 NA 42
54 NA 57
67 16 76
a
growth inhibition value > 50 at 10-5M drug concentration considered as significant. bNA - no cytotoxic activity.
pH 7.4, 10% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin) at 370C in an atmosphere of 5% CO 2 and 95% relative humidity in a CO 2 incubator. The cells at subconfluent stage were harvested from the flask by treatment with trypsin (0.05% trypsin in PBS containing 0.02% EDTA) and suspended in growth medium. Cells with more than 97% viability (trypan blue exclusion) were used for determination of cytotoxicity. An aliquot of 100 µl of 2.0 x 105 cells/ml of SNB-78, 1.5 x 10 5 cells/ml of HOP-62, 1.0 x 10 5 cells/ml of T47D and 1.0 x 10 5 cells/ml of cervix (SiHa) were transferred to a well of 96-well tissue culture plate. The cells were allowed to grow for 24 h at 37 0C in a CO 2 incubator as stated above. Test materials (100 µl) were then added to the wells and cells were further allowed to grow for another 48 h. Suitable blanks and positive controls were also included. Each test was done in triplicate. The cell growth was stopped by gently layering of 50 µl of 50% trichloroacetic acid (TCA). The plates were incubated at 40C for an hour to fix the cells attached to the bottom of the wells. Liquids of all the wells were gently pipette out and discarded. The plates were washed five times with distilled water to remove TCA, growth medium, etc and were air-dried. 100 µl of SRB solution (0.4% in 1% acetic acid) was added to each well and the plates were incubated at room temperature for 30 min. The unbound SRB was quickly removed by washing the wells five times with 1% acetic acid. Plates were air dried, tris-buffer (100 µl of 0.01M, pH 10.4) was added to all the wells and plates were gently
stirred for 5 minutes on a mechanical stirrer. The optical density was recorded on ELISA reader at 540 nm. The growth inhibition value > 50 at 10-5 M drug concentration is considered as significant. In vivo anti-tumoral screening All experiments were conducted following CPCSEA ethical committee guidelines. Closed colony bred Swiss albino male mice [6-7 weeks age, 24 ± 2 gm] were obtained from the animal colony of CNCI, housed in cage and maintained on standard mouse food and tap water ad libitum. EAC and S-180 cells freshly obtained from NCCS were used. Tumor cell suspensions in physiological saline were prepared as described (9) to final concentrations of 5 x 10 6 cells/ml. Mice were inoculated with 106 viable cells/mouse at an injection volume of 0.2 ml on day 0. Mice were divided in two groups as control (untreated) and treated. At least 6 animals were used for a particular group in an experiment. Physiological saline containing 2% Tween 80 (Sigma Inc., USA) was used for drug administration in respective doses through intraperitoneal (i.p.) route to each mouse in different schedules as per Table 2. The drug solutions were prepared daily just prior to the injection. The control groups received an equal volume of vehicle (0.2 ml) on those days. The number of deaths were counted daily during the test. The testing was evaluated by calculating the median survival times (M.S.T.) (10) of drug-treated (T) and control (C) tumorbearing animals and expressed as percent T/C value. A T/C percentage value > 125 is considered as significant.
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Table 2: In vivo screening data a Compound 1
Tumor System
Expt. No.
Dose (mg/kg)
No. of Injections.
Injections days
MST (days)
Survivors > 60 days
T/Cb (%)
S-180
1
50 Control 50 50 50 Control 50 50 Control 50 25 Control 1.0d 0.5 Control 50 Control 50 50 Control 50 50 Control
7 – 6 5 5 – 7 7 – 5 5 – 7 7 – 7 – 6 5 – 7 7 –
1–7 – 1–6 1–5 1,3,5,7,9 – 5–11c 10–16c – 1–5 1–5 – 1-7 1-7 – 1–7 – 1–6 1–5 – 5–11c 10–16c –
>60 20.5 51.0 48.0 53.0 24.0 51.0 43.0 25.5 49.5 >60 21.5 25.0 36.5 19.5 51.0 21.5 48.0 46.5 23.0 43.5 36.5 21.5
4 – 3 2 3 – 2 1 – 2 4 – 1 – 3 – 2 2 – 2 1 –
>292 100 212 200 221 100 200 169 100 230 >279 100 128 187 100 237 100 209 202 100 202 170 100
25 Control
5 –
1-5 –
60.0 23.5
4 -
>240 10
2
3 CCNU
4
Mitonafide
5
1
EAC
6 7 8
CCNU
9
a
n = no. of animals in each group is 6. Mice were inoculated with 106 tumor cells by i.p. route on day 0. Compounds were administered by i.p. route as per schedule. Mortality was observed daily over a period of 60 days [for details see Materials and Methods]. b %T/C value > 125 is considered as significant. cDrug challenge in advanced tumor dRef. 7.
3
H-Thymidine and 3H-Uridine incorporation in S-180 cells in vitro
In vivo toxicological assay Based on the in vivo antitumoral assay, the optimum dose of 50 mg/kg was administered in normal and S180 bearing mice from day 1 to 7. Various parameters were measured sequentially after 48 hr (day 9) for noting immediate effects, after 192 hr (day 15) for intermediate effects and after 336 hr (day 21) for late effects. For haematological studies, blood samples were collected from tail veins or by cardiac puncture from recently sacrificed animals under deep exposure to pentothal sodium for counting erythrocytes, thrombocytes, leukocytes and measuring haemoglobin concentrations (Fig. 1) (11). After removal from the sacrificed anaesthetized animals, whole spleen and femoral marrow cells were collected and processed (9). Serum alkaline phosphatase (SAKP), glutamic oxaloacetic transaminase (SGOT), glutamic pyruvic transaminase (SGPT) and blood urea nitrogen (BUN) levels were measured (11) in the sera obtained from blood samples on day 9, 15 and 21. Abbreviations used for groups of mice: NC - normal control; NT - normal treated; SC - S-180 control; ST S-180 treated. 18
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H-thymidine (specific activity 1.0 mCi/ml) and 3Huridine (specific activity 1.0 mCi/ml) were obtained from Board of Radiation and Isotope Technology, Mumbai, India. Radioactivity was measured in a liquid scintillation counter. The assay was conducted as per the procedure described earlier (12). Briefly tumor cells aspirated from a mouse bearing S-180 for 7 days were washed twice in Hank’s balanced salt solutions and re-suspended in RPMI-1640 medium supplemented with 10% heat inactivated foetal calf serum, streptomycin (100 µg/ml) and penicillin (100 units/ml). Cell suspensions taken in glass tubes were made and adjusted in such a fashion so that the final cell count became 1 × 106/100 µl after the addition of 3H-thymidine or 3H-uridine (activity 10 µCi each) dissolved in 10 µl sterile saline and NitronaphthalNU at 8 µM concentration based on the concentration of Mitonafide reported (4). Same concentration of CCNU was also used. The tubes were incubated at 37ºC for 30 and 60 min. Cell viability assessed by trypan blue dye exclusion test was of the order of 95%. The cells were harvested at 0, 30 and 60 min of incubation and absorbed
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Nitronaphthal-NU as a novel antitumor agent
Hemoglobin
RBC
110
110
95
95
80
80
65
65
50
50 9
15
21
9
21
Platelet
WBC
% of normal control
15
800
250
600
200
400
150
200
100
0
50 9
15
21
9
Bone marrow cellularity
15
21
Splenic cellularity
120
250
100
200
80
150
60
100 50
40 9
15
21
Day
9
15 NT
ST
21 SC
Figure 1. Sequential changes in hematological parameters, femoral bone marrow and splenic cellularities in normal and S-180 bearing mice (n = 6 in each group) after treatment with Nitronaphthal-NU from day 1-7. Parameters were expressed with respect to values [Hemoglobin – 14.5 gm/dl; RBC – 7.7 x 106 /µl; WBC – 8.2 x 103 /µl; Platelet – 6.9 x 105 /µl; Femoral bone marrow cell count – 12.2 x 106 /µl; Splenic cell count – 15.2 x 107 /µl] obtained for normal control mice (n = 40).
onto 25-mm discs of Whatman 3MM filter papers. The dried discs were washed twice with ice-cold 10% TCA followed by with absolute alcohol. The discs were then dried in air, placed in scintillation vials containing scintillation fluid and the radioactivity was counted. Experiments were carried out in triplicate. For background counts, filter papers were soaked with 10 µl of 10 µCi of 3H-thymidine or 3H-uridine, processed as described above and the radioactivity counted. Actual incorporations of the isotopes in the drug-treated groups were calculated by subtracting the
background count from the observed counts. Results were expressed as percentage of the incorporation in the appropriate control without drug (Fig. 2). Statistical analysis Values were recorded as the mean ± S.E.M. Experimental results were analysed by student’s t-test. P< 0.05 was considered as the level of significance for values obtained for treated groups compared with the normal group.
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RNA synthesis
(% control)
Incorportation of precursor
DNA synthesis 100
100
80
80
60
60
40
40
20
20
0
0 0
30
60
0
30
60
Incubation time (min) Mitonafide
CCNU
Compound 1
Figure 2. Effects of compound 1, CCNU and Mitonafide at 8 µM concentration each on the synthesis of DNA and RNA in S-180 tumor cells (For details see Materials and Methods). Results are expressed as a percentage of the incorporation in the appropriate control that contained no drug. P < 0.01 obtained from six experiments.
RESULTS In vitro screening results revealed significant (p<0.01) cytotoxicity of the compound 1 in two cell lines namely SNB-78 CNS and HOP-62 lung as the percentage growth inhibition values were 65 and 67 respectively at 10-5 M drug concentration. CCNU did not exhibit cytotoxicity in any cell line. Mitonafide has exhibited cytotoxic effect in the HOP-62 lung cell line (Table 1). The LD50 value of Nitronaphthal-NU in mice was found to be > 400.0 mg/kg by single i.p. injection. Experiments could not be carried out at higher doses due to its precipitation in the vehicle. The growth responses in S-180 and EAC were assessed following drug treatment in different doses and schedules in vivo. For determining the optimum dose, detailed studies were initially carried out in S-180 (Expt. no. 1-3, Table 2). The optimum dose was found to be 50 mg/kg for the schedule 1-7 day [QD1-7] having the T/Cmax value >292 (Expt. no. 1) (p<0.001). In vivo experiments in EAC with this optimum dose furnished comparable results having T/C value of 237 (Expt. no. 6). CCNU has also demonstrated highly significant (p<0.001) anticancer activity in these tumor systems having long-term survivors (T/C values in S-180 and EAC are >279 and >240 respectively). Mitonafide was simultaneously assessed in S-180 by employing 0.5 mg/kg and 1 mg/kg doses for 1-7 days (7). The optimum dose of Mitonafide was found to be 0.5 mg/kg for the same schedule [QD1-7] having maximum T/C value of 187 (Expt. no. 5, Table 2). To study the haematological changes associated with drug application, four parameters – (a) haemoglobin level (b) erythrocyte count (c) leukocyte 20
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count and (d) platelet counts were determined in the peripheral blood of NT, ST and SC groups. Femoral bone marrow and splenic cellularities were also determined. The detailed data obtained for each parameter in these groups were expressed in Fig. 1 as the percent control of the respective values. From Fig. 1, it was noted that there were about 14 18% decrease in the haemoglobin levels and erythrocyte counts in the NT and ST groups on day 9, which gradually tends to reach the NC value at a later stage. WBC total count results revealed there was no significant decrease or increase in the treated groups. Platelet counts showed that neither thrombocytosis nor thrombocytopenia occurred in the treated groups except slight decrease (10%) in ST group on day 9. It was found that there was slight decrease (18%) in the femoral marrow cell count occurred particularly in the ST group which persisted till day 15 followed by recovery. Hyposplenic cellularity noted on day 9 in ST (decrease by 15%) similarly reached normal count within day 15-21. For evaluating drug-induced hepatotoxicity and nephrotoxicity, SAKP, SGPT, SGOT and BUN values obtained for the treated groups were compared with those of NC mice (respective values are 16.0 IU/l, 6.5 IU/l, 6.6.KA unit and 12.0 mg/dl). It is worth noting that all the values remained within normal range in the treated groups (Data not presented). Since compound 1 has remarkable structural resemblance with Mitonafide, studies were conducted to ascertain whether drug-induced tumor growth inhibition was also due to the inhibitory effect of this compound on nucleic acid synthesis apart from its alkylating property. Hence 3 H-thymidine and 3 Huridine uptake by S-180 cells harvested from untreated mouse were evaluated after treating the cells in vitro.
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Nitronaphthal-NU as a novel antitumor agent
The untreated S-180 cells demonstrated an almost linear pattern of 3H-thymidine and 3H-uridine uptake over a period of 60 min incubation. Simultaneous exposure of tumor cells to compound 1 at the concentration of 8µM resulted in gradual and marked inhibition of 3H-thymidine and 3H-uridine uptake comparable to that of Mitonafide at the same concentration (8µM). After 1 hr incubation time, with respect to control Mitonafide inhibited 99 % of DNA synthesis inhibition while respective values for compound 1 and CCNU were 91 % and 92%. It was also noted that compound 1, CCNU and Mitonafide have exhibited greater inhibitory effect on DNA synthesis compared to RNA synthesis. The respective values for Mitonafide, compound 1, CCNU are as the following 94%, 87% and 88% in respect of RNA synthesis (Fig. 2). This fully corroborates earlier observation (4) that Mitonafide preferentially blocked DNA synthesis than RNA synthesis.
DISCUSSION It is noteworthy that compound 1 has significantly (p<0.001) prolonged the life span of tumor bearing mice in different schedules with 1-4/6 animals having survival rates of > 60 days. In order to simulate a clinical situation where a cancer chemotherapeutic agent is often used at an advanced stage of disease, its efficacy was evaluated in different doses in mice bearing S-180 and EAC for 5 days and 10 days before the drug challenge. It is interesting to note that T/C values of 169 and 170 with 1 cure/6 mice were obtained in the respective groups bearing highly advanced 10 days tumor (Expt. no. 3 and 8, Table 2). It was found that nitronaphthal-NU has shown greater in vivo anticancer activity than Mitonafide and CCNU in mice bearing S-180. Literature survey reveals (13) that tumorigenesis and its progression has been accompanied with the following changes compared to normalcy: (1) gradual decrease in haemoglobin content, erythrocyte count and bone marrow cellularity (2) gradual increase in leucocytes, thrombocytes and splenic cellularity which was observed in SC mice. Different parameters sequentially measured in the ST group reflected almost similar picture of NC mice. Slightly decreased counts in some parameters in the treated groups on day 9 were soon elevated to the normal values within day 21. All these results indicated that compound 1 did not adversely affect haematopoiesis at its optimum dose. It is well known that there are significant elevations in the levels of SAKP and SGPT in liver diseases and damages caused by a number of agents (11). An increase in the SGOT level is observed in patients with
cardiac damage due to myocardial infarction and with liver disorders. An increase in the BUN level is noted in cases of renal diseases and damage (BUN level > 30.0 mg/dl is considered significant for toxicity) (11). Since the values remained within the normal range in treated groups, compound 1 has not displayed hepatotoxicity or nephrotoxicity at its optimum dose. Our presumption that compound 1 would significantly inhibit the synthesis of the DNA and RNA was supported from the data obtained (Fig. 2). From the antitumoral and toxicological assay results, it is concluded that Nitronaphthal-NU possesses promising chemotherapeutic potential.
ACKNOWLEDGEMENTS We wish to express our sincere thanks to Director of CNCI, for constant encouragement, to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for financial assistance [Grant Number: 01(1466)/97/EMR-II] and to Dr. M. F. Brana, Vice Chancellor, Univ. of San Pablo – CEU, Madrid, Spain, for generous supply of Mitonafide.
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