|Year : 2013 | Volume
| Issue : 3 | Page : 442-446
Evaluation of antitumor efficacy and toxicity of novel 6-nitro-2-(3-chloropropyl)-1H-benz[de]isoquinoline-1,3-dione in vivo in mouse
Asama Mukherjee, Sushanta Dutta, Utpal Sanyal
Department of Anticancer Drug Development, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
|Date of Web Publication||8-Oct-2013|
Department of Anticancer Drug Development, Chittaranjan National Cancer Institute, Kolkata, West Bengal
Source of Support: None, Conflict of Interest: None
Aim: This study was aimed to assess the in vivo anti-tumoral potency of the novel 6-nitro-2-(3-chloropropyl)-1H-benz[de]isoquinoline-1,3-dione [Compound 1] that has earlier demonstrated excellent cytotoxicity in 15 out of 17 human tumor cell lines tested.
Materials and Methods: Two murine tumors namely Sarcoma-180 (S-180) and Ehrlich ascites carcinoma (EAC) were used to measure its in vivo anti-tumor activity through the increase in median survival times (MST) of drug treated (T) over untreated control (C) mice. Drug-induced toxicity in respect of hematological parameters, femoral bone marrow and splenic cellularity as well as biochemical parameters and histopathology of liver and kidney were assessed in vivo in normal and S-180 bearing mice sequentially on days 9, 14 and 19 following drug treatment at the optimum dose of 60 mg/kg administered from day 1 to 7.
Results: Results revealed significant tumor regression effects in S-180 and EAC as T/C max values of 138 and 189 were obtained at its optimum dose of 60 mg/kg for QD 1-7 . Toxicity assay indicated no significant cardiotoxicity, hepatotoxicity or nephrotoxicity of the compound in normal and S-180 bearing mice. An initial hyposplenic cellularity and the femoral bone marrow suppression effect observed on day 9 reached normalcy by day 19. HPLC analysis revealed that it has appreciable stability (half-life ~ 3 h) in murine blood plasma in vitro.
Conclusion: Above results justify potential candidature of the compound for further drug development.
Keywords: In vivo assay, naphthalimide based compound, novel antitumor agent, plasma stability, toxicity
|How to cite this article:|
Mukherjee A, Dutta S, Sanyal U. Evaluation of antitumor efficacy and toxicity of novel 6-nitro-2-(3-chloropropyl)-1H-benz[de]isoquinoline-1,3-dione in vivo in mouse. J Can Res Ther 2013;9:442-6
|How to cite this URL:|
Mukherjee A, Dutta S, Sanyal U. Evaluation of antitumor efficacy and toxicity of novel 6-nitro-2-(3-chloropropyl)-1H-benz[de]isoquinoline-1,3-dione in vivo in mouse. J Can Res Ther [serial online] 2013 [cited 2019 Dec 13];9:442-6. Available from: http://www.cancerjournal.net/text.asp?2013/9/3/442/119332
| > Introduction|| |
Small molecules having a naphthalimide [1H-benz(de)isoquinoline-1,3-dione] core have a well-defined place in the current research for chemotherapeutic agents. M.F. Brana et al. rationally developed a number of such molecules containing N-(2,2-dimethylaminoethyl) chain best represented by Mitonafide having 5-nitro group in the aromatic ring and Amonafide having 5-amino group in the aromatic ring [Figure 1] that possess excellent anti-tumor activities. These have undergone Phase I-II clinical trials with limited results. ,
We have recently reported the synthesis, in vitro cytotoxicity and mechanism of action of a series of N-[2-(chloroethyl)]- and N-[3-(chloropropyl)]-naphthalimides as a new class of antineoplastic agents.  Among them 6-nitro-2-(3-chloropropyl)-1H-benz[de]isoquinoline-1,3-dione [compound 1 , [Figure 1], which revealed excellent cytotoxicity in 15 out of 17 human tumor cell lines tested, was subjected to extensive screening in two murine tumors namely S-180 and EAC in Swiss Albino mice in vivo. Further, its toxicity in normal and S-180 bearing mice was assessed at its optimum dose. The stability of the compound in murine blood plasma was also determined quantitatively using HPLC and half-life was calculated from the calibration curve.Present report describes all these aspects.
| > Materials and Methods|| |
Chemicals and reagents
6-nitro-2-(3-chloropropyl)-1H-benz[de]isoquinoline-1,3-dione (98% purity by HPLC) was prepared as reported.  Anticancer drug Endoxan (Cyclophosphamide) was purchased from Sigma-Aldrich Com., USA. Mitonafide was received as a generous gift from Prof. M.F. Brana, University of San Pablo-CEU, Madrid, Spain. Other solvents procured from Merck, India were either analytical or HPLC grade.
Swiss albino male mice of about 6-8 weeks of age and weighing 24 ± 2gm were used for the experiments. Animals were randomly bred in the vivarium of CNCI, housed in cage and maintained on standard mouse food and tap water ad libitum. All in vivo experiments were conducted following the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals).
In vivo screening
Maximum tolerated dose of the compound was determined by administration to normal Swiss male mouse at the dose range up to 400 mg/kg body weight. EAC and S-180 cells freshly obtained from NCCS, Pune were used. Tumor cell suspensions in physiological saline were prepared to final concentrations of 5 × 10 6 cells/ml. Mice were inoculated with 10 6 viable cells per 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 administration of the compound in respective doses through intraperitoneal (i.p.) route to each mouse in different schedules as per [Table 1]. 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 was counted daily during the test and body weights of mice were checked at an interval of 5 days starting from day 0. The testing was evaluated by calculating the median survival times (M.S.T.)  of drug-treated (T) and control (C) tumor-bearing animals and expressed as percent T/C value. A T/C percentage value > 125 is considered as significant. Experimental compound Mitonafide and Endoxan were included as positive controls.
In vivo toxicological assay
The optimum dose of 60 mg/kg of compound 1 was administered from day 1 to 7 to normal and S-180 bearing mice after inoculation with 1 ×10 6 cells on day 0. The control groups received only vehicle on those days. After sacrificing mice from the respective groups (6 animals in each group), various parameters were measured sequentially after 48 h (day 9) for noting immediate effects, after 168 h (day 14) for intermediate effects and after 288 h (day 19) for late effects. For hematological studies, blood samples were collected by cardiac puncture from recently sacrificed animals under deep exposure to pentothal sodium. Erythrocytes, thrombocytes, leukocytes were counted and hemoglobin concentrations were measured in those samples [Figure 2]. After removal from the sacrificed anaesthetized animals, whole spleen and femoral marrow cells were collected and processed. Biochemical parameters as SGOT, SGPT, SAKP, BUN and creatinine were measured , in the sera obtained from blood samples on day 9, 14 and 19 [Figure 3] using test kits procured from Span Diagnostics Ltd, Surat, India. The detailed procedure as described by the manufacturer was followed.Liver and kidney of sacrificed mice of all the four groups were collected on those days and fixed in neutral buffered formalin. These were processed by standard method to prepare slides of tissues by hematoxylin and eosin (H and E) staining. The slides were viewed under light microscope [Olympus BX50] for histopathological studies [Figure 4].
|Figure 2: Sequential changes in hematological parameters, femoral bone marrow and splenic cellularity in normal and S-180 bearing mice after treatment with compound 1 from day 1-7.|
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|Figure 3: Sequential changes in biochemical parameters in normal and S-180 bearing mice after treatment with compound 1 from day 1-7.|
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|Figure 4: Photomicrographs of hematoxylin and eosin [H and E] stained sections of liver and kidney of Swiss albino mouse.(a) NC liver, (b) SC liver (day 14), (c) ST liver (day 9), (d) ST liver (day 14), (e) ST liver(day 19),(f) NT kidney (day 9) [a, b, d, e magnification× 200; c, f magnification× 400].|
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Waters HPLC system at ambient temperature (M-501 / M-510 solvent delivery pump, M-481 UV-Vis variable - wavelength detector set at 255nm / 696 Photodiode Array detector, U6K injector, nova-pak C18 steel column was used. Isocratic mobile phase containing methanol and buffer (15 mM ammonium acetate in 0.02% formic acid) were passed through 0.50 and 0.45 μm Millipore filters respectively and mixed in desired proportion (3:1, v/v), followed by degassing under stirring in a low-vacuum oil-free pump was eluted at a flow rate of 0.5 ml/min at ambient temperature. Data analysis was done in Empower Build 1154 Software.
Stock solution of compound 1 (400 μg/ml) was prepared in HPLC grade methanol. The solution was stable for 15 days when kept at 0-4°C. The stock solution was serially diluted by adding methanol to yield final concentrations in the range of 40 - 0.40 μg /ml. Each of these solutions was injected (injection volume = 10 μl) in nova-pak column and the areas so obtained were plotted against the amount of the compound. The limit of quantification (LOQ) and limit of detection (LOD) were determined by gradually reducing the injection volume of the solution over at least six successive determinations.
Stability and half-life determination in murine blood plasma
Blood samples were collected from normal healthy Swiss albino mice by cardiac puncture in micro centrifuge tubes containing the anticoagulant EDTA. Platelet free blood plasma was obtained by centrifuging first at 4000 rpm for 15 min and then at 10,000 rpm for 30 min and stored at -80°C.
Plasma was spiked with known amount of compounds so that three solutions having 10, 25 and 50 mM concentrations were obtained and analyzed as described below. The concentration of 25 mM was found as the optimum one for obtaining results with precision and accuracy.
Thus to 1 ml of plasma, 10 μl of compound 1 in DMSO was added in such a fashion to get the effective concentration of 25 μM. This was incubated in a 37°C water bath with shaking up to 8 h. Aliquots of 80 μl were drawn at time intervals of 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 8 h from the reaction mixture. Cold methanol (160 μl) was added to the aliquots for protein precipitation followed by centrifugation at 1500 rpm for 10 min. 100 μl of supernatant was drawn and 20 μl of the sample was injected thrice to obtain the mean area. The concentration of unchanged compound 1 in plasma was calculated from the standard curve at different time intervals and its half-life was determined [Figure 5].
Values were recorded as the mean ± S.E.M. (standard error mean) of three experiments. Experimental results were analyzed by student's t-test. P < 0.05 was considered as the level of significance for values obtained for treated groups compared with the control group.
Abbreviations used: NC, normal control; NT, normal treated; SC, S-180 control; ST, S-180 treated; SGOT, Serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase; SAKP, serum alkaline phosphatase; BUN, blood urea nitrogen; DMSO, Dimethyl sulphoxide; EDTA, Ethylenediaminetetraacetate.
| > Results|| |
In vivo screening
The maximum tolerated dose of compound 1 in mice was found to be > 400 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 [Experiment no. 1, [Table 1] at different doses and schedules. A T/C max value of 138 was obtained for compound in S-180 at the dose of 60 mg/kg for QD 1-7 [Experiment no.1, [Table 1]. Thus the compound has exhibited marginally significant activity in S-180. However in EAC, at the same dose and schedule it has shown much better activity [% T/C max 189, Experiment no. 3, [Table 1]. It also revealed marginal activity (T/C max 130, Experiment no. 4) in EAC for QD 5-11 schedule.
It was found that under similar treatment schedule (QD 1-7 ), Mitonafide administered at the optimum dose of 0.5 mg/kg has displayed a T/C max value of 187 in S-180 [Experiment no. 2, [Table 1]. Endoxan revealed T/C max values of 143 and 138 [Experiment no.2 and 5, [Table 1] respectively in S-180 and EAC tumor systems. Thus compound 1 has shown comparable activity with Endoxan in S-180 and greater activity in EAC.
In the tumor growth inhibitory study in S-180, after the application of compound 1 at its optimum dose, total ascites cells were counted and ascetic fluid volumes measured. Results showed a significant inhibition (84%) in S-180 tumor growth on day 9 after its application. Thereafter gradual growth of the tumor was noted and on day 19 there was ~ 20% inhibition. Similar observation was noted for ascites fluid formation (figures not presented). This was also reflected in the body weights of treated and control mice.
In vivo toxicological assay
From [Figure 2], it was noted that there were about 18-20% and 12-18% decreases in the hemoglobin levels and erythrocyte counts in the NT and ST groups on day 9, which gradually tended to reach the NC value at a later stage. On the 9 th day; WBC counts decreased by nearly 15% in both the treated groups; followed by elevation to NC value later on. Platelet counts showed that neither thrombocytosis nor thrombocytopenia occurred in the treated groups except slight decrease (15%) in NT group on day 9. It was found that there were about 18-19% depression in the femoral marrow cell counts in the treated groups initially that were gradually recovered by day 19. Hypospleenic cellularity (decrease by 42-48%) more pronounced in the NT groups was particularly noted on day 9. Gradual increase in the spleenic cellularity was observed in these groups thereafter and the normal counts were attended within day 19. The average spleen weights recorded in the NT and ST groups, however, did not exhibit any distinct change.
For evaluating drug-induced hepatotoxicity and nephrotoxicity, SGOT, SGPT, SAKP, BUN and creatinine values obtained for the treated groups were compared with those of NC mice (respective values are 17.4 IU/l, 15.2 IU/l, 9.1 KA unit, 10.3 mg/dl and 1.3 mg/dl) [Figure 3] (Figure for creatinine not presented). It is worth noting that all the values remained within normal range in the treated groups.
H and E - stained section of liver of NC mouse showed normal architecture having a distinct central vein within a lobule and a portal area consisting of interlobular branch of portal vein, bile duct, branch of a hepatic artery and interlobular septum [Figure 4]a. Histology of liver of SC mouse (14 day) showed hyperactive Kupffer cells and lymphocyte infiltration in areas adjacent to central vein as well as hepatic parenchyma [Figure 4]b. Photomicrographs of liver of NT mice treated with title compound showed normal architecture of hepatic lobes and hepatocytes on day 9, 14 and 19 suggesting no significant hepatotoxicity in this group (Figure not presented). On day 9, hyperactive Kupffer cells and lymphocyte infiltration on the liver parenchyma were observed in ST group suggesting mild inflammatory changes in liver [Figure 4]c. On day 14, few necrotic cells along with some regenerated hepatocytes were seen in the liver section [Figure 4]d. Infiltration of lymphocytes was observed in lower numbers in ST compared to SC group. Enhanced activity of Kupffer cells was observed on day 14 compared to day 9 in ST group.On day 19, Kupffer cell activity returned to pre-treatment level and there was no trace of lymphocyte infiltration in ST group [Figure 4]e. Hepatocytes were normal and regularly arranged within the hepatic parenchyma, suggesting hepatic recovery.
Photomicrographs of kidney of tumor control (SC) and treated (NT and ST) mice showed normal histology. A section of NT kidney represented in [Figure 4]f shows that the cortex contained well-disposed renal corpuscles (i.e. intact Bowman's capsule and glomerulus), convoluted tubules and medullary rays. The inner medulla region contained the Henle's loop and the collecting tubules. Thus the compound [or its metabolite(s)] at the optimum dose does not possess nephrotoxicity.
The LOD and LOQ determined for the title compound were found to be 0.4 μg/ml and 4.0 μg/ml respectively. The desired compound was eluted at 8.9 min under the chromatographic condition used. It was found that the compound was considerably stable in plasma and could be detected over the entire period of study (8 h). No interfering peaks were found in the extracted blood plasma. The chromatograms showed that the peak area of the test compound gradually decreased as the incubation time increased. The concentration of unchanged compound plotted against time was shown in [Figure 5]. From the figure it appears that after about 3 h the compound degraded to 50% of the initial concentration, which denotes the half-life of the compound in plasma.
| > Discussion|| |
The usual dose-limiting toxicity of Mitonafide and other naphthalimide compounds in general is the myelosuppression as well as gastrointestinal, hematologic and neurologic toxicities. Literature survey reveals  that tumorigenesis and its progression have been accompanied with the following changes compared to normalcy: 1) gradual decrease in hemoglobin content, RBC count and bone marrow cellularity 2) gradual increase in WBC, platelet and splenic cellularity which was actually observed in SC mice. Mild insignificant decreases were noted in hematological parameters in the treated groups on day 9 that tended to reach normal values subsequently [Figure 2]. A multitude of pathological changes occur during the progression of tumor as well as its inhibition by chemotherapeutic agents. These are expected to be reflected in the biochemical and histological parameters of the host system, particularly in organs like the liver where detoxification occurs or in the kidneys that help in elimination. 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.  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 and creatinine levels is noted in cases of renal diseases and damage.  Since the values remained within the normal range in treated groups, compound has not displayed significant hepatotoxicity or nephrotoxicity at its optimum dose.
Histopathological analysis of liver in drug treated mouse revealed mild inflammatory reaction initially which was recovered at later stage. This is a common observation in liver sections facing an external drug assault. 
Drug stability in plasma is a matter of great concern in drug development. Drug candidates undergoing rapid degradation in plasma generally have undesirable pharmacokinetic parameters and pose analytical challenges. For potential candidates, plasma instability will complicate validation of the bio-analytical assays for the unstable pharmaceutical components or their metabolites in plasma samples. Our study has shown considerable stability of the compound in murine blood plasma, which is a pre-requisite for a potential drug candidate.
All the above data support potential candidature of the title compound for further drug development.
| > Acknowledgements|| |
We express our sincere thanks to the Council of Scientific and Industrial Research, New Delhi, India, for financial assistance [Grant Number: 01(1791)/02/EMR-II to U.S.], to Dr. Jaydip Biswas, Director, CNCI, for encouragement and to Dr. Manas Ranjan Ray, Head, Department of Experimental Hematology, CNCI, for histopathological data analysis.
| > References|| |
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]