Sibabrata Mukhopadhyay

Natural Products: Promising Resources for Cancer Drug Discovery

Natural products are important sources of anti-cancer lead molecules. Many successful anti-cancer drugs are natural products or their analogues. Many more are under clinical trials. The present review focuses on chemopreventive and anti-cancer activities of polar and non-polar extracts, semi purified fractions and pure molecules from terrestrial plants of India reported between 2005 and 2010 emphasizing possible mechanisms of action of pure molecules.

Targeting of liposomal andrographolide to L. donovani-infected macrophages in vivo.

Despite the rapid development in medicinal and pharmaceutical technology, the targeting of drugs to phagocytic cells in macrophage-related diseases still remains a major unsolved problem. By using the mannosyl-fucosyl receptors on macrophages, attempts were made to target antileishmanial drugs encapsulated in mannosylated or fucosylated liposomes to treat experimental leishmaniasis in the hamster model. Mannosylated liposomes were found to be more potent in delivering antileishmanial drugs to phagocytic cells. Liposomes loaded with an indigenous drug, andrographolide, a labdane diterpenoid isolated from Indian medicinal plant Andrographis paniculata, were prepared and tested against experimental leishmaniasis in a hamster model. Mannosylated liposomes loaded with the drug were found to be most potent in reducing the parasitic burden in the spleen as well as in reducing the hepatic and renal toxicity. In addition, mannosylated drug-loaded liposome-treated animals showed a normal blood picture and splenic tissue histoarchitecture when compared with those treated with free drug or regular liposomal drug. Such a drug-vehicle formulation may be considered for clinical trials.Despite the rapid development in medicinal and pharmaceutical technology, the targeting of drugs to phagocytic cells in macrophage-related diseases still remains a major unsolved problem. By using the mannosyl-fucosyl receptors on macrophages, attempts were made to target antileishmanial drugs encapsulated in mannosylated or fucosylated liposomes to treat experimental leishmaniasis in the hamster model. Mannosylated liposomes were found to be more potent in delivering antileishmanial drugs to phagocytic cells. Liposomes loaded with an indigenous drug, andrographolide, a labdane diterpenoid isolated from Indian medicinal plant Andrographis paniculata, were prepared and tested against experimental leishmaniasis in a hamster model. Mannosylated liposomes loaded with the drug were found to be most potent in reducing the parasitic burden in the spleen as well as in reducing the hepatic and renal toxicity. In addition, mannosylated drug-loaded liposome-treated animals showed a normal blood picture and splenic tissue histoarchitecture when compared with those treated with free drug or regular liposomal drug. Such a drug-vehicle formulation may be considered for clinical trials.

Harmine: evaluation of its antileishmanial properties in various vesicular delivery systems.

Harmine, a beta-carboline amine alkaloid isolated from Peganum harmala, was tested for its antileishmanial properties both in vitro and in vivo. In vitro antileishmanial activity of harmine was encouraging and prompted us to confirm the activity in vivo in hamster models. Harmine was tested both in free form and in different vesicular forms viz. liposomes, niosomes and nanoparticles. The different vesicles were prepared by the published protocols. The percent intercalation of harmine in liposomes, niosomes and nanoparticles was found to be 65, 60 and 20, respectively, when determined at 325 nm (∈M=2.33 × 104 M-1 cm-1). At an equivalent dose of 1.5 mg/kg body weight, injected subcutaneously (SC) for a total of six doses in 15 days, harmine was found to reduce spleen parasite load by approximately 40, 60, 70 and 80%, respectively in free, liposomal, niosomal and nanoparticular forms. An inverse relationship could be established between the efficacy in the lowering of spleen parasite load and the size of the vesicles. Specific biochemical tests related to normal liver and kidney functions revealed that the toxicity of the drug was reduced in the vesicular forms in the same order as their efficacy and the same was confirmed by the histopathological studies of splenic sections. Cell cycle analysis studies using flow cytometry suggested that although harmine interferes in the cell division stage, it does not induce apoptosis in Leishmania donovani promastigotes. The results using Confocal Microscopy supported that the cell death could be attributed to necrosis due to non-specific membrane damage. Even then, because of its appreciable efficacy in destroying intracellular parasites as well as non-hepatotoxic and non-nephrotoxic nature, harmine, in the vesicular forms, may be considered for clinical application in humans.

Quercetin interferes with iron metabolism in Leishmania donovani and targets ribonucleotide reductase to exert leishmanicidal activity

OBJECTIVES The possibility of developing antileishmanial drugs was evaluated by intervention in the parasites iron metabolism, utilizing quercetin (Qr) under in vivo conditions, and identifying the target of this lipophilic metal chelator against Leishmania donovani. METHODS Interaction between Qr and serum albumin (SA) was studied by using the intrinsic fluorescence of Qr as a probe. The effect of treatment with Qr and SA on the proliferation of amastigotes was determined by evaluating splenic parasite load. Disintegration of parasites in response to combination treatment was assessed from ultrastructural analysis using a transmission electron microscope. Quenching of the tyrosyl radical of ribonucleotide reductase (RR) in treated amastigotes was detected by an electron paramagnetic resonance study. RESULTS Treatment with a combination of Qr and SA increased bioavailability of the flavonoid and proved to be of major advantage in promoting the effectiveness of Qr towards the repression of splenic parasite load from 75%, P < 0.01 to 95%, P < 0.002. Qr-mediated down-regulation of RR (P < 0.05), catalysing the rate-limiting step of DNA synthesis in the pathogens, could be related to the deprivation of the enzyme of iron which in turn destabilized the critical tyrosyl radical required for its catalysing activity. CONCLUSIONS Results have implications for improved leishmanicidal action of Qr in combination with SA targeting RR and suggest future drug design based on interference with the parasites iron metabolism under in vivo conditions.

Quercetin: Critical Evaluation as an Antileishmanial Agent In Vivo in Hamsters Using Different Vesicular Delivery Modes

Chemotherapy is still a major challenge for in vivo drug targeting to macrophages. Toxicity remains the major obstacle for the most potent drugs already known in the therapy of leishmaniasis. Thus, new drugs and new delivery systems are sought. By using different vesicular delivery modes e.g. liposomes, niosomes, microspheres and nanoparticles, attempts have been made to deliver an indigenous antileishmanial compound, quercetin, to treat experimental leishmaniasis in the hamster model so as to increase its efficacy as well as to reduce the toxicity. At equivalent quercetin concentration, the nanocapsulated quercetin was found to be the most potent in reducing the parasite burden in the spleen as well as in reducing hepatotoxcity and renaltoxicity compared to free drug or drug in other vesicular forms. An inverse relationship between the efficacy and the size of the vesicles was established. Such a drug vehicle formulation especially in the nanocapsulated form may be considered for clinical trials.

Novel betulin derivatives as antileishmanial agents with mode of action targeting type IB DNA topoisomerase.

Toward developing antileishmanial agents with mode of action targeted to DNA topoisomerases of Leishmania donovani, we have synthesized a large number of derivatives of betulin. The compound, a natural triterpene isolated from the cork layer of Betula spp. plants exhibits several pharmacological properties. Three compounds (disuccinyl betulin, diglutaryl dihydrobetulin, and disuccinyl dihydrobetulin) inhibit growth of the parasite as well as relaxation activity of the enzyme type IB topoisomerase [Leishmania donovani topoisomerase I (LdTOP1LS)] of the parasite. Mechanistic studies suggest that these compounds interact with the enzyme in a reversible manner. The stoichiometry of these compounds binding to LdTOP1LS is 1:1 (mole/mole) with a dissociation constant on the order of ∼10−6 M. Unlike CPT, these compounds do not stabilize the cleavage complex; rather, they abrogate the covalent complex formation. In processive mode of relaxation assay condition, these compounds slow down the strand rotation event, which ultimately affects the relaxation of supercoiled DNA. It is noteworthy that these compounds reduce the intracellular parasite burden in macrophages infected with wild-type L. donovani as well as with sodium antimony gluconate resistant parasite (GE1). Taken together, our data suggest that these betulin derivatives can be exploited as potential drug candidates against threatening drug resistant leishmaniasis.

Role of oxidation‐triggered activation of JNK and p38 MAPK in black tea polyphenols induced apoptotic death of A375 cells

Theaflavins (TF) and thearubigins (TR) are the major polyphenols of black tea. Our previous study revealed that TF‐ and TR‐induced apoptosis of human malignant melanoma cells (A375) is executed via a mitochondria‐mediated pathway. In our present study we observed the role of the three most important MAPK (ERK, JNK, and p38) in TF‐ and TR‐induced apoptosis. TF and TR treatment of A375 cells led to sustained activation of JNK and p38 MAPK but not ERK, suggesting that JNK and p38 are the effector molecules in this polyphenol‐induced cell death. This idea was further supported by subsequent studies in which JNK and p38 activation was inhibited by specific inhibitors. Significant inhibition was found in TF‐ and TR‐treated A375 cell death pretreated with JNK‐ or p38‐specific inhibitors only. Further, we have found that TF and TR treatment induces a time‐dependent increase in intracellular reactive oxygen species generation in A375 cells. Interestingly, treatment with the antioxidant N‐acetyl cystein inhibits TF‐ and TR‐induced JNK and p38 activation as well as induction of cell death in A375 cells. We also provide evidence demonstrating the critical role of apoptosis signal‐regulating kinase 1 in TF‐ and TR‐induced apoptosis in A375 cells. Taken together our results strongly suggest that TF and TR induce apoptotic death of A375 cells through apoptosis signal‐regulating kinase 1, MAPK kinase, and the JNK–p38 cascade, which is triggered by N‐acetyl cystein intracellular oxidative stress. (Cancer Sci 2009; 100: 1971–1978)

Swertane triterpenoids from Swertia chirata

Abstract Two novel triterpenes belonging to swertane skeleton, besides gammacer-16-en-3β-ol and 21α H -hop- 22(29)-en-3β-ol, of rare occurrence have been isolated from Swertia chirata , along with some common triterpenoids. Their structures were established on the basis of spectral and chemical evidence.

Comparative antimutagenic and anticancer activity of three fractions of black tea polyphenols thearubigins.

Antimutagenic and anticancer effects of black tea polyphenols theaflavins (TF) and thearubigins (TR) have previously been reported. TR is a complex mixture of polyphenols. In this study, our interest was to fractionate TR and to study the antimutagenic and anticancer activities of the fractions. Three fractions of TR, namely TR-1, TR-2, and TR-3, were isolated by chromatographic processes. Antimutagenic activity of these 3 fractions was carried out on 4 Salmonella strains by Ames assay. Anticancer activity was studied on human leukemic cells U937. Our findings clearly indicated antimutagenic and anticancer activities of the TR-1, TR-2, and TR-3 fractions on Salmonella strains and on U937 cells, respectively. However, all 3 fractions, at or below 100 μg/ml dose, did not show any significant toxic effects on the normal human cells (peripheral blood mononuclear cells). TR-2 was found to be the most active fraction among the 3. Flow cytometric and confocal microscopic studies further indicate that apoptosis induction could be an important mechanism behind the anticancer effects of these fractions. To our knowledge, this study is the first attempt to describe the antimutagenic and anticancer activity of TR fractions, and it also suggests that TR-2 is the most active component of TR.

Targeting of Liposomal Flavonoid to Liver in Combating Hepatocellular Oxidative Damage

The efficacy of mannosylated liposome formulations with Quercetin (QC, a flavonoid antioxidant isolated from indigenous origin) has been tested in vivo against carbon tetrachloride(CCl 4) -induced liver oxidative damage in rats. Single subcutaneous injection of CCl 4 (40% v/v in olive oil; 1 ml/kg) induces the generation of toxic oxygen radicals and results in hepatocellular damage. The increased serum enzyme levels (glutamate pyruvate transaminase, alkaline phosphatase) and hepatocellular conjugated diene levels by CCl 4 induction were significantly lowered due to pretreatment with mannosylated liposomal QC (MLQ) (0.5 ml liposomal suspension containing 0.27 mg QC), whereas the same amount of free QC was found to be ineffective. In addition, the effectiveness of MLQ on CCl 4 -induced acute liver damage also was evaluated by tissue histopathological examination. Damage produced by CCl 4 in liver reverted to normal with pretreatment of MLQ.