Somenath Banerjee

Renal handling of amino acid 99mtechnetium chelates

Four amino acids--alanine, 2,3-diaminopropionic acid, cystine, and cystein--and also one diamine, ethylenediamine, were chelated with 99m-technetium (99mTc), and their renal excretion patterns were studied in rabbits in the presence and absence of two renal tubular transport inhibitors, probenecid and 2,4-dinitrophenol. From the depression of renal excretion for the first three amino acid chelates, in the presence of the inhibitors, a renal tubular excretory pathway of elimination was suggested for these compounds. The renal excretions of 99mTc-cystein and 99mTc-ethylenediamine however, remained undepressed under similar experimental conditions. An explanation of these observations was forwarded from the possible chemical structures of these chelates.

Exploring the anti-inflammatory activity of a novel 2-phenylquinazoline analog with protection against inflammatory injury

Inflammation is a protective immune response against harmful stimuli whose long time continuation results in host disease. Quinazolinones are nitrogen containing heterocyclic compounds with wide spectrum of biological activities. The anticancer effect of a 3-(arylideneamino)-phenylquinazoline-4(3H)-one derivative was reported earlier. The anti-inflammatory effect of these quinazolinone derivatives has now been examined in endotoxin stimulated macrophages and in different in vivo models of inflammation by measuring the proinflammatory cytokines (TNF-α, IL-1β and IL-6), mediators NO and NF-κB (by ELISA and western blot), and translocation of the nuclear factor kB (by immunocytochemical analysis). To elucidate the in vivo effect, mice endotoxin model was and the various levels of edema, inflammatory pain and vascular permeability were studied. One of the quinazolinone derivatives showed significant anti-inflammatory activity in stimulated macrophage cells by inhibiting the expression of TNF-α, IL-1β, IL-6, iNOS, COX-2, p-IκB and NF-κBp65. Significant (P<0.01) improvement was observed in the mortality of endotoxemic mice. The carrageenan and formalin-induced paw edema thicknesses were found to be reduced significantly (P<0.01) along with the reduction of pain, vascular permeability and edema induced by complete Freunds adjuvant (P<0.01). These findings indicate that 3-(arylideneamino)-phenylquinazoline-4(3H)-one derivative as a potential anti-inflammatory agent.

Anomalies in hepatobiliary excretion of technetium-99m-MAG3 preparations

Technetium-99m-MAG3 is accepted as a renal tubular function agent. However, sporadic liver and gall bladder visualisation during its clinical use is clearly a disadvantage. HPLC-purified 99mTc-MAG3 samples exhibited appreciable hepatobiliary uptake (7%), and an elevated level of such uptake was observed in unpurified kit preparations, which was stated to be associated with the excretory property of the radiolabeled kit impurities. To verify this we attempted to quantitate the hepatobiliary uptake of the kit preparations with that of its radiolabeled components. The contribution of each component toward hepatobiliary uptake of the sample was calculated from their abundance in the chelate mixture and the individual biodistribution of the isolated components. However, the anticipated hepatobiliary uptake of different preparations of 99mTc-MAG3 calculated in this way was always lower than that of the experimental value determined directly. Further work is needed to explain the anomaly.

Cysteine, a chelating moiety for synthesis of technetium-99m radiopharmaceuticals—Part IV. Benzyl cysteine and derivatives

To explore the possibility of utilizing cysteine derivatives for technetium-99m radiopharmaceutical preparation with clinical potential, we synthesized two benzyl substituted cysteine compounds, namely, S-benzyl cysteine 1 and cysteine benzyl ester 3. It was expected, from our previous studies on benzoyl cysteines, that the above two ligands after chelation with 99mTc would be excreted by the hepatobiliary pathway. Although for 99mTc-3 the above expectation was realized, 99mTc-1 behaved in a most unexpected way by affixing itself with kidney and selecting the renal tubular secretory pathway for its excretion. It is anticipated that the affinity of 99mTc-1 for kidney is due to its interaction with the kidney sulphhydryl group and it also formed an adduct with other sulphydryl containing compounds like thiophenol. In terms of the kidney-to-background ratio, 99mTc-1 showed some superiority over other kidney structure agents, like 99mTc-dimercaptosuccinic acid and 99mTc-glucoheptanoic acid and, therefore, the chelate (99mTc-1) may have the potential to replace the above two radiopharmaceuticals in clinical use.

Leishmanial lipid affords protection against oxidative stress induced hepatic injury by regulating inflammatory mediators and confining apoptosis progress.

Persistence of liver injury alters the internal milieu, promotes deregulation of inflammatory factors, and leads to dysplastic lesions like fibrosis, cirrhosis to hepatocellular carcinoma. Our previous study revealed that leishmanial lipid (pLLD) exerts potential anti-inflammatory activity in sepsis associated hepatic injury. We now show that pLLD gives protection against chemical induced hepatotoxicity in murine system. The beneficial effect of treatment with pLLD on such hepatic injury in mice was analyzed using different assays including ELISA, FACS, western blot and immunohistochemical analysis. pLLD significantly suppressed serum enzymes and rectified the histopathological alteration to induce the antioxidant level in CCl4 intoxicated liver. Levels of several growth factors including TGF-β, HGF, and EGF were significantly improved in serum and hepatic tissue with consequent reduction of caspase activities and expressions of Bad, Bax, p53, and NF-κBp65. Moreover, pLLD modulated inflammatory responses by decreasing the production of several cytokines and chemokines, thus preventing the infiltration of immune cells to the damaged area. It accelerated the repair process in liver damage with modulation of signalling cascade via alteration of apoptotic factors. Our experimental approaches suggest that pLLD effectively prevents liver injury mainly through down regulation of oxidative stress and inflammatory response towards anti-apoptotic changes.

99Tcm-cystine, a renal function and imaging agent: a comparative study in dog with 131I-hippurate and 99Tcm-glucoheptonate to evaluate its functional and imaging characteristics

99Tcm-cystine, which has been proposed as a renal radiopharmaceutical for evaluating renal morphology and function in a single experiment, is compared with 131I-orthoiodohippurate (OIH) with respect to its renal clearance and extraction parameters and with 99Tcm-glucoheptonate (GHA) regarding its imaging characteristics. In spite of its comparable renal accumulation with 131I-OIH, its clearance (10.1 ± 1.0 ml min-1 kg-1) was lower than that of 131I-OIH (21.5 ± 0.9 ml min-1 kg-1) but was higher than that of 125I-iothalamate (5.4 ± 0.6 ml min-1 kg-1). Extraction efficiencies of 99Tcm-cystine, 131I-OIH and 125I-iothalamate were 39 ± 5, 64 ± 4 and 27 ± 3, respectively. The glomerular filtration components of 99Tcm-cystine and 131I-OIH were 26 and 16% of their respective clearances. In probenecid-treated animals the clearance of both agents was affected to a similar extent and fell to half of their respective control values, whereas tubular secretory components were found to be 19 and 31% of the controls. The kidney images obtained with 99Tcm-cystine were superior to those obtained with 99Tcm-GHA at different time points. Therefore, considering both renal function and imaging properties of 99Tcm-cystine it appears that this radiopharmaceutical offers some definite advantages over the currently available renal agents and commands further study.

Lipid isolated from a Leishmania donovani strain reduces Escherichia coli induced sepsis in mice through inhibition of inflammatory responses.

Sepsis is the reflection of systemic immune response that manifests in the sequential inflammatory process in presence of infection. This may occur as a result of gram-negative bacterial sepsis including Escherichia coli infection that gives rise to excessive production of inflammatory mediators and causes severe tissue injuries. We have reported earlier that the lipid of attenuated Leishmania donovani suppresses the inflammatory responses in arthritis patients. Using heat killed E. coli stimulated macrophages, we have now investigated the effect of leishmanial total lipid (LTL) isolated from Leishmania donovani (MHO/IN/1978/UR6) for amelioration of the inflammatory mediators and transcriptional factor with suppression of TLR4-CD14 expression. To evaluate the in vivo effect, E. coli induced murine sepsis model was used focusing on the changes in different parameter(s) of lung injury caused by sepsis, namely, edema, vascular permeability, and pathophysiology, and the status of different cytokine-chemokine(s) and adhesion molecule(s). Due to the effect of LTL, E. coli induced inflammatory cytokine-chemokine(s) levels were significantly reduced in serum and bronchoalveolar lavage fluid simultaneously. LTL also improved the lung injury and suppressed the cell adhesion molecules in lung tissue. These findings indicate that LTL may prove to be a potential anti-inflammatory agent and provide protection against gram-negative bacterial sepsis with pulmonary impairment.

Technetium-99m amino acid chelates: Correlation of their physico-chemical and physiological parameters. Part I.

Ten alpha-aminocarboxylic acids were chelated with 99mTc and purified by sephadex gel chromatography. Their hepatobiliary and renal excretion patterns were determined in rats. It was observed that lipophilicity of these chelates is the only determinant in governing their hepatobiliary excretion, whereas their renal excretion is dependent on some other factors in addition to lipophilicity. Depression of renal excretion in presence of probenecid indicated an interaction of renal tubular transport enzymes with these chelates, which was explained from their hexacoordinated dioxotechnetium (V) chelate structure (II).

Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

Background/Aims: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy.

Leishmanial sphingolipid induces apoptosis in Sarcoma 180 cancer cells through regulation of tumour growth via angiogenic switchover.

Sphingolipids are membrane and intracellular lipids that typically modulate cellular processes to cause cell death. Exogenous administration of sphingolipids may cause restriction of tumour growth and several alternative strategies are being used to control the cell growth. The microbes, their cellular component(s) or metabolites like DHA, EPA and also FTY720 have been employed as new therapeutic entities to regulate the disease condition. The therapeutic efficacy of lipids from Leishmania donovani in rheumatoid arthritis and also in sepsis condition associated with inflammatory diseases is well established. In this study, we explored the apoptotic effect of LSPL-1 (leishmanial sphingolipid-1) in Sarcoma 180 cells towards the regulation of tumour growth. The study using a panel of cancer cell lines revealed that LSPL-1 induces cell death in Sarcoma 180. The apoptotic changes were assessed by annexin exposure and DNA content analysis using flow cytometry. LSPL-1 appears to activate several pro- and anti-apoptotic molecules through reactive oxygen species (ROS) generation and also caspase activation, as determined by Western blot and ELISA analyses. Simultaneously, it may improve the survival rate of mice bearing tumour induced by Sarcoma 180 cells, with pathological changes. LSPL-1 may also suppress the cancer-associated inflammatory responses with the expression of matrix metalloproteinase having inhibitory role. It may regulate several angiogenic factors including VEGF, Ang-2 and CD34 in angiogenic events generated in Sarcoma 180 cell-induced tumour. These studies underline the significance of anti-neoplastic potential of LSPL-1 through apoptosis induction and abrogation of angiogenic responses in Sarcoma 180 cell-associated tumour.