INTERNATIONAL JOURNAL OF PHARMACEUTICAL … 1163.pdfSN-38 has a potency to induce SSB that is 1000 times stronger than that ... (HIV) [8], apparently as a result of the inhibition

Research Article CODEN: IJPRNK ISSN: 2277-8713 Debpratim Chakraborty, IJPRBS, 2015; Volume 4(5): 300-314 IJPRBS

Available Online at www.ijprbs.com 300

ANTI TUMOR ACTIVITY OF CPT-11 AND ITS ACTIVE METABOLITE SN-38

DEBPRATIM CHAKRABORTY1, ANANYA SAHA2, RAJESH KUMAR KHATICK3, DR. SHAMPA

CHAKRABORTY

Pursuing M. Pharm from Jadavpur University, West bengal.

Accepted Date: 13/10/2015; Published Date: 27/10/2015

Abstract: Now a day cancer is a hot trend of researches. Cancer rates could further increase by 50% to 15 million new cases in the year 2020. The conventional marketed medicines are not capable to cure it, that’s why scientist are going for new drug discovery. Camptothecin (CPT) is a monoterpene indole alkaloid produce potent anti cancer activity but due to its several toxicity, a derivative of CPT known as CPT-11 and its active metabolite SN-38 is taken granted. Both are topoisomerase I inhibitor. CPT-11 showed higher anti-tumor activity than Adriamycin, 5-fluro uracil in rat and mouse. On Leukemia cell SN-38 caused the strongest inhibition of the relaxation of SV40 DNA by topoisomerase I prepared from P388 cells, followed by CPT-11. In this experiment, the respective IC50 of SN-38 and CPT-11 were 0.74 and >1000 µM and the IC25 were 0.20 µM and 0.72 mM respectively. (SN-38 show about 3600-fold stronger activity than CPT-11). SN-38 has a potency to induce SSB that is 1000 times stronger than that of CPT-11. In another study on Human Colon Carcinoma CPT-11 Enhances the Antitumor Activity of LZ-huTRAIL in Vivo. Administration of CPT-11 alone resulted in a dose-dependent inhibition of HT-29 tumor growth, with six doses of CPT-11 at 25 mg/kg/dose resulting in ;50% reduction in tumor size, and 50 mg/kg/dose resulting in; 75% reduction. As seen combination of LZ-huTRAIL (250 or 500 mg/day) plus 25 mg/kg CPT-11 resulted in a much greater tumor growth inhibition than that seen with either agent alone. These results indicate that CPT-11 itself possesses a potent anti tumor effect and that SN-

38 plays an essential role in the mechanism of action of CPT-11.

Keywords: CPT-11, SN-38, Leukemia, Human colon carcinoma

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Debpratim Chakraborty, IJPRBS, 2015; Volume 4(5): 300-314



INTRODUCTION

Now a day every one of these of this world is concern about cancer though reports say

that cancer rates could further increase by 50% to 15 million new cases in the year 2020. The

conventional drugs and procedure are not enough for total cure of malignancy. So newer drug

discovery is very much important for mankind as soon as possible. After a long study scientist

said that Camptothecin has potent activity on tumor but its several toxicity they have to turn

their mind towards the semi synthetic derivative of Camptothecin (CPT), named as CPT-11(7

ethyl-10-[4-(1-Piperidino) -1-piperidino] carboxyloxycamptothecin). Here we are going to

discuss about CPT-11 and its anti tumor activity.

Cancer: Cancers are a large family of diseases that involve abnormal cell growth with the

potential to invade or spread to other parts of the body.[1][2] They form a subset of neoplasms.

A neoplasm or tumor is a group of cells that have undergone unregulated growth, and will

often form a mass or lump, but may be distributed diffusely.[3][4]

Six characteristics of cancer have been proposed:

Selfsufficiency in growth signaling

insensitivity to antigrowth signals

evasion of apoptosis

enabling of a limitless replicative potential

induction and sustainment of angiogenesis

activation of metastasis and invasion of tissue.[5]

The progression from normal cells to cells that can form a discernible mass to outright cancer

involves multiple steps known as malignant progression.

Prevelence: GENEVA, 3 APRIL 2003 - Cancer rates could further increase by 50% to 15 million

new cases in the year 2020, according to the World Cancer Report, the most comprehensive

global examination of the disease to date. In the year 2000, malignant tumors were responsible

for 12 per cent of the nearly 56 million death worldwide from all causes. In many countries,

more than a quarter of deaths are attributable to cancer. In 2000, 5.3 million men and 4.7

million women developed a malignant tumor and altogether 6.2 million died from the disease.

The report also reveals that cancer has emerged as a major public health problem in developing

countries, matching its effect in industrialized nations.



Tobacco, the case for primary prevention: Tobacco consumption remains the most important

avoidable cancer risk. In the 20th century, approximately 100 million people died world-wide

from tobacco-associated diseases (cancer, chronic lung disease, cardiovascular disease and

stroke). Half of regular smokers are killed by the habit. One quarter of smokers will die

prematurely during middle age (35 to 69 years). The lung cancer risk for regular smokers as

compared to non-smokers (relative risk, RR) is between 20 and 30 fold. Roughly 90 per cent of

lung cancers in both men and women are attributable to cigarette smoking. For bladder and

renal pelvis, the RR is five-six but this means that more than 50 per cent of cases are caused by

smoking. Involuntary (passive) tobacco smoke is carcinogenic and may increase the lung cancer

risk by 20 per cent.

Infection and cancer: intervention is key: In developing countries, up to 23 per cent of

malignancies are caused by infectious agents, including hepatitis B and C virus (liver cancer),

human papilloma viruses (cervical and ano-genital cancers), and Helicobacter pylori (stomach

cancer). In developed countries, cancers caused by chronic infections only amount to

approximately 8 per cent of all malignancies. Today, more than 80 per cent of all cervical cancer

deaths occur in developing countries.

Cancer by the Numbers: Lung cancer is the most common cancer worldwide, accounting for 1.2

million new cases annually; followed by cancer of the breast, just over 1 million cases;

colorectal, 940,000; stomach, 870,000; liver, 560,000; cervical, 470,000; esophageal, 410,000;

head and neck, 390,000; bladder, 330,000; malignant non-Hodgkin lymphomas, 290,000;

leukemia, 250,000; prostate and testicular, 250,000; pancreatic, 216,000; ovarian, 190,000;

kidney, 190,000; endometrial, 188,000; nervous system, 175,000; melanoma, 133,000; thyroid,

123,000; pharynx, 65,000; and Hodgkin disease, 62,000 cases.[6] The three leading cancer

killers are different than the three most common forms, with lung cancer responsible for 17.8

per cent of all cancer deaths, stomach, 10.4 per cent and liver, 8.8 per cent. Industrial nations

with the highest overall cancer rates include: U.S.A, Italy, Australia, Germany, The Netherlands,

Canada and France. Developing countries with the lowest cancer were in Northern Africa

Southern and Eastern Asia.

Camptothecin (CPT) and its derivatite CPT 11: Camptothecin (CPT) is a monoterpene indole

alkaloid produced by the Chinese tree Camptotheca acuminata Decne (Nyssaceae) and first

isolated in 1966 by Wall and coworkers. CPT accumulation was observed in more than 90% of

the histological sections of all of the organs examined (leaf, stem and root). CPT is known for its

remarkable anti-cancer activity, which results from its ability to inhibit the eukaryotic DNA

topoisomerase I [7]. It also inhibits retroviruses such as the human immunodeficiency virus

(HIV) [8], apparently as a result of the inhibition of Tat-mediated transcription [9]. CPT not used

clinically, however because of severe toxicity. Recently CPT-11 7 ethyl-10-[4-(1-Piperidino) -1-



piperidino] carboxyloxycamptothecin, was semi synthetic as a CPT derivative which has potent

anti tumor activity, low toxicity and high water solubility. CPT-11 showed higher anti-tumor

activity than Adriamycin, 5-fluro uracil in rat and mouse. It was shown to be a topoisomerase I

inhibitor. CPT-11 is an ester prodrug and is enzymetically hydrolyzed to the active metabolite.

7-ethyl-10-hydroxycamptothecin (SN-38). Tsuji et al. reported that CPT-11 was hydrolyzed by

serum carboxylesterase.

Extraction of CPT: Naturally occurring camptothecins (CPT) are important sources of

chemotherapeutic agents for clinical treatment of cancer. Extraction of CPT from Camptotheca

acuminate trees remains to be a cost effective way in the supply equation compared with a

total synthesis. This study conducted a series of experiments to determine efficient solvent for

the maximal extraction of CPT and its two derivatives, hydroxycamptothecin (HCPT) and

methoxycamptothecin, from seeds and leaves of C. acuminata. Methanol as an extraction

solvent demonstrated in seeds a significantly higher recovery of these three alkaloids than

dichloromethane and acetone. Methanol concentrations at 70% in water resulted in maximum

extraction of all the three alkaloids regardless of the type of plant materials. However, other

strengths of methanol, lower or higher, either decreased the extracting power or showed no

improvement in the extraction. Seed extract contained all the three alkaloids whereas leaf

extract was absent of HCPT. A stable ratio of the three alkaloids was discovered but it was

dependent upon seed or leaf extract of C. acuminata, which with various compositions can be

produced. Ecological and medicinal implications of the leaf and seed extract characterized with

different chemical compositions are discussed.

MECHANISM OF ACTION OF CAMPTOTHECIN: Earlier studies have shown that camptothecin is

a strong inhibitor of both DNA and RNA synthesis [10]. DNA synthesis is rapidly inhibited by

camptothecin and only partially recovered upon drug removal [11]. At high camptothecin

concentrations, DNA synthesis is irreversibly inhibited and S-phase cells cannot progress into

the G2 phase of the cell cycle [12]. Inhibition of RNA synthesis by camptothecin appears to be

more severe for high molecular weight RNAs (e.g., rRNA and heterogeneous nuclear RNA) than

for low molecular weight RNAs (4S and 5S RNAs). The inhibition is rapid and reversible. In

addition to its inhibition of nucleic acid synthesis, camptothecin has also been found to induce

"reversible" DNA strand breaks in Hela cells [13]. It is not clear whether the induction of DNA

strand breaks is related to the inhibition of nucleic acid synthesis. Recently, mammalian DNA

topoisomerase I has been suggested as a possible intracellular target of camptothecin (14).

Using purified calf thymus DNA topoisomerase I, it has been demonstrated that camptothecin

can inhibit the enzyme by trapping an enzyme-DNA intermediate, termed the "cleavable

complex" (14). Although the exact chemical nature of this cleavable complex is still unclear, it

has been shown that treatment of drug-induced cleavable complexes with protein denaturants



results in single-strand DNA breaks and the concomitant covalent linking of a topoisomerase I

polypeptide to the 3' phosphoryl end of each strand break (14). Treatment of the cleavable

complexes with high concentrations of salts (e.g., 0.5 MNaCl) prior to the addition of protein

denaturants results in rapid "reversal" of the cleavage reaction.

Activity of CPT-11 and SN-38 on Leukemia cell:The inhibition rate of topoisomerase I activity

was calculated as : [1 –(FIri-FIr)/(FIre-FIr)]x 100 (%)

Where FIri and FIre are the ratios of relaxed DNA to total DNA treated with topoisomerase I in

the presence and absence of a test compound, respectively. FIr is the proportion of relaxed DNA

in untreated DNA. The IC50 of each test compound was estimated from the dose-response

curve.

The intracellular content of SN-38 was determined as

[SN-38]FT - [SN-38]C

Where [SN-38]FT and [SN-38]C are the amounts of SN-38 in cells processed with and without

freezing and thawing, respectively.

Result of treatment of CPT-11 and SN-38 on Topoisomerase I: In a study the effects of CPT-11

and SN-38 on Activity of Topoisomerase I. SN-38 caused the strongest inhibition of the

relaxation of SV40 DNA by topoisomerase I prepared from P388 cells, followed by CPT and then

CPT-11 (Fig. 1). In this experiment, the respective IC50 of SN-38, CPT, and CPT-11 were 0.74, 2.3,

and >1000 µM, as listed in Table 1. For comparison of the inhibitory activities of SN-38 and CPT-

11, the IC25 value for topoisomerase I of P388 was calculated similarly to that of the IC 50. This

value of CPT-11 was 0.72 mM, whereas that of SN-38 was 0.20 µM (about 3600-fold stronger

than CPT-11).

CPT-11 dose dependently shifted the position of relaxed DNA in the direction of nicked DNA, as

shown in Fig. 1. SN- 38 and CPT showed no effect on the position of relaxed DNA in the

experiments described above. CPT-11, SN-38, and CPT dose-dependently inhibited DNA

synthesis. The synthesis was decreased to 20% of the control at 100 µM of CPT-11. At a

concentration of 1 µM, SN- 38 and CPT reduced the synthesis to 12 and 21% of the control,

respectively. The inhibitory effect of each compound on RNA synthesis was less than that on

DNA synthesis. No inhibition was observed in protein synthesis after the treatment with any of

the compounds. Respective IC50 values of CPT-11, SN-38, and CPT in DNA synthesis were 19,

0.077, and 0.18 µM, as listed in Table 2.



Table 1. Inhibitory activity of SN-38, CPT, and CPT- II against relaxation of supercoiled DNA by

topoisomerase 1

Agent IC50µM

P388 Ehrlich SN-38 0.74(1.0) 1.9(1.0)

CPT 2.3(3.1) 7.5(3.9) CPT-11 >1000 >1000

Table 2. Inhibitory effects of SN-38, CPT, and CPT-ll on DNA and RNA synthesis in P388 cells

Agent IC50µM

DNA RNA

SN-38 0.74(1.0) 1.9(1.0)

CPT 2.3(3.1) 7.5(3.9)

CPT-11 >1000 >1000

The time-dependent effects of each test compound on DNA synthesis and cellular thymidine

uptake are measured. The inhibitory effect of CPT-11 against DNA synthesis was time

independent, although those of SN-38 and of CPT were time dependent. Moreover, the 20-min

treatment with CPT-11 reduced the cellular thymidine uptake to 67 and 25% at 10 and 100 µM,

respectively. This reduction also occurred in a time independent manner. The cellular uptake

was not inhibited by the 60-min treatment with 1 µM of SN-38 or CPT, whereas DNA synthesis

was strongly inhibited. DNA Strand Breaks by CPT-ll and SN-38. After a 1-h treatment, CPT-11

caused SSB much less frequently than SN- 38 and ADM, as demonstrated in Fig. 2. The ability of

CPT- 11 above 400 µM to produce SSB was saturated at about 400 rad-equivalents (Fig. 4A). SSB

frequencies induced by 0.1 µM SN-38 and by 100 µM CPT-11 were 74±2 and 115±22 rad-

equivalents, respectively. A similar frequency (118 rad-equivalents) was induced by l µM ADM.

ADM also induced DSB at 2 µM, but no obvious DSB were detected after treatments with either

SN-38 at 1µM or CPT-11 at 1 mM (data not shown). The relationship between SSB frequency

and DSB frequency is measured which reveals that few or no SSB arising from DSB are included

in the apparent SSB induced by SN-38 or CPT-11.[15]

Relationship between SSB and SN-38: Compares the effects of the 1-h treatment with 100

MM CPT-11 and that with 0.1 MMSN-38 on the frequency of SSB and on the intracellular

content of SN-38. No significant differences in either parameter were observed between these



two treatments. These results indicate that SN-38 has a potency to induce SSB that is 1000

times stronger than that of CPT-11 and that most SSB induced by CPT-11 are due to SN-38.

Discussion: CPT-11 shows potent antitumor activities in antitumor tests in vivo.[16] SN-38, a

metabolite of this compound, possesses a much higher cytotoxicity against tumor cells in vitro,

although it seems to be less effective in vivo, in comparison with CPT-11. CPT, the mother

compound of CPT-11, is known as a specific inhibitor of topoisomerase I [17]. The inhibition of

this enzyme is thought to be responsible for the cytotoxicity of CPT-11 on the molecular level.

CPT-11 at 100 µM is contaminated with only 5 nM of SN-38, and the culture medium increased

the concentration of SN-38 to less than 10 nM under the conditions under which SSB were

tested. However, the treatment with CPT-11 at 100µM induced a similar frequency of SSB and

gave an intracellular content of SN-38 similar to those after treatment with SN-38 at 0.1µM.

These observations indicate that SN-38 was produced from CPT-11 in cells and that the SSB

induced by CPT-11 were principally due to this SN-38. The inhibitory effects of CPT-11 on DNA

synthesis and on RNA synthesis were approximately 250 and 50 times less than those of SN-38,

respectively (Table 2). The inhibitory effects of CPT-11 were time independent, whereas those

of the other compounds were time dependent. Moreover, CPT-11 induced coincident time-

independent inhibitions of the cellular uptakes of thymidine and uridine, which were not

induced by either SN-38 or CPT. When the cells were treated with SN-38 or CPT, the

radioactivity of the acid-soluble fraction increased and compensated for the decrease in that of

the acid-insoluble fraction, but it was unchanged after treatment with CPT-11. These results

suggest that CPT-11 suppresses independently of time the regulatory mechanism(s) of the

transport of nucleic acid precursors and that this suppression relates to the apparent inhibition

of nucleic acid synthesis.

Even if CPT-11 possesses unexpected action(s) on nucleic acid synthesis, this compound itself

appears to have a marginal antiproliferative effect, because the concentrations required to

show such actions are much higher (>250 times) than those at which SN-38 affects

topoisomerase I and DNA synthesis and induces DNA strand breaks. Furthermore, as stated

above, it is believed that topoisomerase I inhibition and SSB after treatment with CPT-11

depend principally on SN-38. Therefore, at the intracellular level, it appears that SN-38 plays a

dominant role in the antitumor effect caused by CPT-11.[18]

Activity of CPT-11 and SN-38 Human Colon Carcinoma Tumors::

Treatment of Tumor-bearing Mice with LZ-huTRAIL: Female CB.17 (SCID) mice were pretreated

24 h before tumor challenge with a single injection (100 µl, i.p.) of purified asialo GM-1

antibody. Mice were injected s.c. with 3x105 human colon carcinoma cells, and treatment

began 3, 10, or 17 days after tumor implantation, as noted. Treatments with TBS or LZ-huTRAIL



were administered by i.p. injection, and CPT-11 was administered i.v. All dilutions of LZ-huTRAIL

and CPT-11 were made with TBS. For the 10- and 17-day established tumor study, changes in

tumor size were calculated as follows:

[(Tumor size post treatment) - (Tumor size at day 10 or 17)/(Tumor size at day 10 or 17)] x

100%.[19]

RESULTS::

Human Colon Carcinoma Tumors Show a Differential Sensitivity to LZ-huTRAIL in Vivo. :

Previous work has shown that human colon carcinoma cell lines are differentially sensitive to

LZ-huTRAIL induced apoptosis in vitro.3 To determine the antitumor activity of LZhuTRAIL in

vivo, tumors derived from human colon carcinoma xenografts were analyzed for their

sensitivity to LZ-huTRAIL (Fig. 3). Beginning 3 days after tumor challenge, mice were treated

with LZhuTRAIL (500 or 1000 mg) or a control solution (TBS) for 14 days. Consistent with the in

vitro findings, tumors derived from COLO 205 and HCT-15 cells were very sensitive to LZ-

huTRAIL treatment (Fig. 3A). The growth rate for both tumors was significantly reduced in the

LZhuTRAIL- treated groups compared with that of control mice. Treatment of COLO 205 tumor

bearing mice with 500 mg/day LZ-huTRAIL resulted in a 90% incidence of tumor formation 6

weeks after tumor challenge, whereas treatment with 1000 mg/day LZ-huTRAIL resulted in a

30% incidence of tumor formation (P = 0.0002, using a generalized linear model). For HCT-15

tumor-bearing mice, the number of tumor-positive animals at 6 weeks was also significantly

less (60%; P = 0.03) after treatment with 500 mg/day of LZ-huTRAIL. In contrast, tumors derived

from the HT-29 and SW620 cell lines showed less sensitivity to LZ-huTRAIL (Fig. 3B).

Camptothecin-11 Enhances LZ-huTRAIL Cytotoxic Activity in Vitro.: Treatment of colon

carcinoma lines with the transcription inhibitor actinomycin D enhances the cytotoxic activity of

LZhuTRAIL in vitro and converts the LZ-huTRAIL-resistant cell lines to LZ-huTRAIL-sensitive cell

lines.3 similar results have been shown using metabolic inhibitors on human melanoma lines

(4). These findings suggest that the antitumor activity of LZ-huTRAIL might be enhanced in vivo

by combining it with chemotherapeutic agents that are capable of disrupting a transformed

cell’s metabolism or mitotic activity. To first test this on the colon carcinoma lines in vitro, a

variety of chemotherapeutic agents were assayed for their ability to synergize with LZ -huTRAIL.

Combining LZ-huTRAIL with cisplatin, 5-FU, mitomycin, etoposide, or Adriamycin did not result

in any enhancement of cytotoxic activity in vitro (data not shown). In contrast,

Camptothecin was found to be a potent cytotoxic agent both alone and in combination with LZ -

huTRAIL. Camptothecin is a topoisomerase I inhibitor that has antitumor activity in vitro and in

vivo (8 –11). The addition of camptothecin (1mg/ml) to colon carcinoma cell lines in vitro

reduced the cell viability of all four cell lines by 40–60% within 24 h (Fig. 2). Incubation for



48 h resulted in a complete loss of cell viability (data not shown).

Combining camptothecin with LZ-huTRAIL converted the LZhuTRAIL- resistant cell lines, HT-29

and SW620, into LZ-huTRAILsensitive cell lines. Likewise, the LZ-huTRAIL-sensitive lines, COLO

205 and HCT-15, became more sensitive to LZ-huTRAIL-induced apoptosis in combination with

camptothecin. These results are similar to those seen with the metabolic inhibitors actinomycin

D and cyclohexamide and confirm that a chemotherapeutic agent has the potential to enhance

the cytotoxic activity of LZ-huTRAIL.[19]

CPT-11 Enhances the Antitumor Activity of LZ-huTRAIL in Vivo.: The synergism observed with

LZ-huTRAIL plus camptothecin in vitro suggested that a similar combination in vivo might

enhance the antitumor activity of LZ-huTRAIL. To study this, LZ-huTRAIL was combined with

CPT-11, a water-soluble analogue of camptothecin. CPT-11 has a broad range of activity against

a variety of human tumors in vivo, including several human colon carcinomas [20-25]. Analysis

of CPT-11 pharmacokinetics has shown that it is converted into its active form, SN38, in mouse

serum and then cleared within a few hours [26]. Consistent with this observation, multiple low

doses of CPT-11 are more effective than a single high dose [27]. To test the combination

therapy of LZ-huTRAIL plus CPT-11 in vivo, LZ-huTRAIL was administered as described previously

(Fig. 1), and CPT-11 was administered by i.v. injection six times during the LZ huTRAIL treatment

period. In these experiments, we focused on the LZ-huTRAIL-resistant HT-29 and LZhuTRAIL-

sensitive COLO 205 tumors. Consistent with the previous analysis (Fig. 3B), treatment of HT-29

tumors with LZ-huTRAIL alone slowed their growth slightly but did not result in any tumor

regressions (Fig. 3A). Administration of CPT-11 alone resulted in a dose-dependent inhibition of

HT-29 tumor growth, with six doses of CPT-11 at 25 mg/kg/dose resulting in ;50% reduction in

tumor size, and six doses of CPT-11 at 50 mg/kg/dose resulting in; 75% reduction. The

combination of CPT-11 plus LZ-huTRAIL resulted in an additional inhibition of tumor growth, but

the difference was not significantly greater than that observed with 25 or 50 mg/kg CPT-11

alone (P = 0.204 and 0.262, respectively). Thus, it appears that the treatment of HT-29 tumors

with a combination of CPT-11 and LZ-huTRAIL results in an additive enhancement of antitumor

activity. LZ-huTRAIL plus 50 mg/kg CPT-11 did dramatically inhibit tumor formation during and

shortly after the treatment period. Control animals and those treated with LZ-huTRAIL alone

were 100% positive for measurable tumors within 7 days of tumor challenge. Likewise, 90–

100% of the animals treated with 50 mg/kg CPT-11 alone were tumor positive within the first 4

weeks. In contrast, LZ-huTRAIL plus CPT-11-treated animals were 70% tumor positive by the

first week but had no measurable tumors at 3 weeks (4 days after treatment). However, these

animals were not completely tumor free, and by 5 weeks, all of them had measurable tumors,

albeit significantly smaller than those in untreated animals (Fig. 3A). Thus, treatment with LZ-

huTRAIL and high-dose CPT-11 could induce a transient regression of LZ-huTRAIL-resistant HT-



29 tumors, but tumor growth resumed with the cessation of treatment. Results of the LZ-

huTRAIL-sensitive COLO 205 tumor treated with LZ-huTRAIL and CPT-11 are shown in Fig. 4. The

administration of either LZ-huTRAIL (500 mg) or CPT-11 (50 mg/kg) alone significantly inhibited

the growth of COLO 205 tumors (P = 0.003 and 0.001, respectively) and induced tumor

regression in 5 of 10 mice and 6 of 9 mice, respectively (Fig. 4A). The combination of LZhuTRAIL

and CPT-11 was even more effective, with all of the treated animals being tumor free 6 weeks

after tumor challenge. To determine whether a lower dose of CPT-11 could synergize with LZ-

huTRAIL, CPT-11 at 25 mg/kg was combined with 250 or 500 mg of LZ-huTRAIL (Fig. 4B). The

administration of LZ-huTRAIL (250 or 500 mg/day) or 25 mg/kg CPT-11 alone resulted in tumor

growth inhibition, but with all treatments, 90% of mice were tumor positive 6 weeks after

tumor challenge. As seen with the 50 mg/kg CPT-11 dose, the combination of LZ-huTRAIL (250

or 500 mg/day) plus 25 mg/kg CPT-11 resulted in a much greater tumor growth inhibition than

that seen with either agent alone. Moreover, 5 of 10 animals treated with 250 mg/day LZ-

huTRAIL and CPT-11 (P = 0.0041) were tumor free, and 18 of 19 animals treated with 500

mg/day LZ-huTRAIL plus CPT-11 (P = 0.0001) were tumor free 6 weeks after tumor challenge.

These results demonstrate that multiple dosing of CPT-11 at 25 mg/kg is well tolerated (only 2%

mortality) and can still synergize with LZ-huTRAIL at two different concentrations and induce

tumor regression in the majority of treated animals. The treatment of COLO 205 tumor-bearing

mice with LZhuTRAIL and/or CPT-11 resulted in many tumor-free animals after 6 weeks. To

determine whether the tumors were completely eliminated, the animals were examined 9 and

12 weeks after tumor challenge (Table 3). The tumor-free animals treated with only CPT-11,

250 mg/day LZ-huTRAIL, or 250 mg/day LZ-huTRAIL plus low-dose. CPT-11 (25 mg/kg) all

developed measurable tumors within 12 weeks. In contrast, the majority of animals treated

with 500 mg/day LZ-huTRAIL, alone or in combination with CPT-11, remained tumor free at 12

weeks. These results suggest that high-dose LZ-huTRAIL (500 mg/day) treatment, alone and in

combination with CPT-11, can induce the complete elimination of COLO 205 tumors.

Table 3. Treatment of COLO 205 tumor-bearing mice with TRAIL (500 mg) or TRAIL (500 mg)

plus CPT-11 results in long-term tumor regression.

Agent Treated mice/surviving

mice

Tumor free mice

6 wks 9 wks 12 wks

CPT-11(25mg/kg) 20/20 1 0 0 CP-11 (50mg/kg) 10/9 6 1 0

TRAIL (250 mg) 10/10 1 0 0 TRAIL (250 mg)+25 mg/kg CPT-11 10/10 5 3 0

TRAIL (500 mg) 20/20 6 4 4

TRAIL (500 mg)+25 mg/kg CPT-11 20/19 18 18 17 TRAIL (500 mg)+50 mg/kg CPT-11 10/7 7 7 7



LZ-huTRAIL plus CPT-11 Induces Regression of Established LZ-huTRAIL-sensitive Tumors: The

treatment protocol used in the previous experiments started LZ-huTRAIL and/or CPT-11

treatment 3 days after tumor challenge. To analyze whether LZ-huTRAIL and

CPT-11 could inhibit the growth of more established tumors, treatments were started at either

10 or 17 days after implantation of COLO 205 cells. At 10 days after tumor challenge, LZ -

huTRAIL (500 mg/day) was administered from day 10–23 (14 days), and six injections of

CPT-11 were given at 25 or 40 mg/kg during the LZ-huTRAIL treatment period. The combination

of LZ-huTRAIL and CPT-11, either 25 or 40 mg/kg, induced the elimination of tumors in 40% and

90% of the mice, respectively (P=0.0033 and 0.0001, respectively). In addition, the tumors in

the remaining mice regressed in size between 50–75% compared with the size of the starting

tumors at day 10 and the size also measured after 17 days. LZ-huTRAIL (500 mg/day) was

administered from day 17–30 (14 days), and CPT-11 was given at 40 mg/kg. In contrast,

treatment with CPT-11 alone induced a 30% regression and 50% regression in tumor size by 6

weeks in comparison to the average starting tumor size at day 17. However, the tumors began

to increase in size after the sixth week, and none of the treatment groups demonstrated any

complete tumor regressions.

DISCUSSION: In this report, we have analyzed four human colon carcinoma cell lines for their

sensitivity to the cytotoxic activity of TRAIL in vivo. Previous work using LZ-huTRAIL verified that

this recombinant form of the molecule retains its activity in vivo [29]. However, it has been

observed that the resistance to TRAIL-induced apoptosis can be overcome in vitro by treating

the cells with metabolic inhibitors [30]. This led us to examine a variety of chemotherapeutic

agents for their ability to enhance TRAIL induced tumor apoptosis. Although cisplatin,

mitomycin, and 5-FU did show a dose-dependent cytotoxicity alone, none showed any synergy

with TRAIL in vitro (data not shown). Treating tumor-bearing mice with TRAIL plus CPT-11, a

water soluble derivative of camptothecin, resulted in a dramatic enhancement of the antitumor

activity of TRAIL. Treatment of 3- or 10-day established COLO 205 tumors with TRAIL and CPT-

11 resulted in both a dose dependent reduction in tumor growth rate and the elimination of

tumors in many of the treated animals. Analysis of these animals for 12 weeks confirmed that

this treatment resulted in tumor-free animals. In contrast, none of the tumors allowed to

establish for 17 days before treatment were eliminated, but a transient shrinkage (50%) of

tumor mass was observed. TRAIL plus high-dose CPT-11 reduced the size of HT-29 tumors .85%

compared with the untreated controls and induced a transient tumor regression after

treatment. However, all animals were eventually tumor positive 6 weeks after tumor challenge.



CONCLUSION:

From the above studies we can conclude that CPT has a potent anti-cancer activity, which

results from its ability to inhibit the eukaryotic DNA topoisomerase-I but due to several toxicity

recently scientists go through a semi synthetic product of CPT known as CPT-11(prodrug) and

its active metabolite SN-38. The activity of cpt-11 and SN-38 is very much prominent over

leukemia and colon carcinoma.In case of leukemia CPT-11, SN-38, and CPT dose-dependently

inhibited DNA synthesis. The synthesis was decreased to 20% of the control at 100 µM of CPT-

11. At a concentration of 1 µM, SN- 38 and CPT reduced the synthesis to 12 and 21% of the

control, respectively. In other hand in case of colon cancer, treating tumor-bearing mice with

TRAIL plus CPT-11, a water soluble derivative of camptothecin, resulted in a dramatic

enhancement of the antitumor activity of TRAIL. In both cases the SN-38 is approximately 3600

fold more active than that of cpt-11. From other studies we may conclude that SN-38is more

potent that the conventional drugs like cisplatin, 5-FU, mitomycin, etoposide and Adriamycin ,

lastly for CPT-11 AND SN-38, less toxicity(ADR) also carry an advantage .



ACKNOWLEDGEMENT:

No work is ever the outcome of single individual’s efforts. This work is also no exception. I wish

to acknowledge the assistance I have received from various people. I am very much thankful to

Dr. Mahananda Sarkar(M.pharm, Ph.D), Mr. Gouranga Barman(M.pharm) Mr. Pritam Goswami

(Final Year Medical student) and also departmental teachers of Jadavpur University.

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INTERNATIONAL JOURNAL OF PHARMACEUTICAL … 1163.pdfSN-38 has a potency to induce SSB that is 1000 times stronger than that ... (HIV) [8], apparently as a result of the inhibition