Kaushiki Mazumdar

New Patentable Use of an Old Neuroleptic Compound Thioridazine to Combat Tuberculosis: A Gene Regulation Perspective

Use of the old antipsychotic phenothiazine thioridazine (THZ) for therapy of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) infection is now being seriously considered. It is reported that THZ primarily acts on enzymes involved in fatty acid metabolism and membrane proteins, particularly efflux pumps, as well as oxidoreductases and proteins involved in aerobic respiration that overlap with a number of conventional antituberculous drugs. It targets the products of the Rv3160c-Rv3161c operon, which are perhaps required for the detoxification of THZ, members of the sigma factor SigB regulon that play a crucial role in protecting the pathogen against cell envelope damage, and Rv2745c, a transcription factor that regulates ATP-dependent proteolysis. Some of these genes have been shown to be essential for the survival or persistence of Mycobacterium tuberculosis in the infected host. Since THZ targets multiple pathways, including those involved in cell wall processes and respiratory chain components, it may serve as a model for multi-target drug development, as well as constitute a highly potent addition to a combination of antituberculous drug regimens. The discussion of some of the patents relevant to thioridazine to combat tuberculosis is also included in the present manuscript.

Antimicrobial potentiality of a new non-antibiotic : the cardiovascular drug oxyfedrine hydrochloride

Ten cardiovascular drugs, having diverse pharmacological action, were screened for possible antimicrobial property against known eight sensitive bacteria, belonging to Gram positive and Gram negative types. Although five drugs failed to show antimicrobial activity and three had moderate antimicrobial action, oxyfedrine HCl and dobutamine were seen to possess pronounced antimicrobial property. Oxyfedrine was further tested in vitro against 471 strains of bacteria from two Gram positive and fourteen Gram negative genera. The minimum inhibitory concentration (MIC) of oxyfedrine was determined by agar dilution method, which ranged from 50-200 microg/ml in most of the strains, while some strains were inhibited at even lower concentrations. In animal experiments, this compound was capable of offering significant protection to Swiss strain of white mice, challenged with 50 median lethal dose (MLD) of a virulent strain of Salmonella typhimurium at concentrations of 15, 30 and 60 microg/mouse. The in vivo results were highly significant according to chi-square test.

Antimycobacterial activity of the antiinflammatory agent diclofenac sodium, and its synergism with streptomycin

Diclofenac sodium, an antiinflammatory agent, exhibited remarkable inhibitory action against both drug sensitive and drug resistant clinical isolates of Mycobacterium tuberculosis, as well as other mycobacteria. This drug was tested in vitro against 45 different strains of mycobacteria, most of which were inhibited by the drug at 10-25 µg/ml concentration. When tested in vivo, diclofenac, injected at 10 µg/g body weight of a Swiss strain of white mice, could significantly protect them when challenged with 50 median lethal dose of M. tuberculosis H37 Rv 102. According to c2 test, the in vivo data were highly significant (p<0.01). Diclofenac was further tested for synergism with the conventional antimycobacterial drug streptomycin against M. smegmatis 798. When compared with their individual effects, synergism was found to be statistically significant (p<0.05). By the checkerboard assessment procedure, the fractional inhibitory concentration index of this combination was found to be 0.37, confirming synergism.

The anti-inflammatory drug Diclofenac retains anti-listerial activity in vivo.

Aims:  The interactions between nonsteroidal anti‐inflammatory drugs (NSAID) and Listeria monocytogenes have not been sufficiently documented to date. The aim of this study was to investigate the possible effects of Diclofenac (Dc) in a murine listerial infection model.

TLR4-independent and PKR-dependent interleukin 1 receptor antagonist expression upon LPS stimulation.

Dendritic cells (DCs) induce innate immune responses by recognizing bacterial LPS through TLR4 receptor complexes. In this study, we compared gene expression profiles of TLR4 knockout (TLR4(neg)) DCs and wild type (TLR4(pos)) DCs after stimulating with LPS. We found that the expression of various inflammatory genes by LPS were TLR4-independent. Among them, interleukin 1 receptor antagonist (IL-1rn) was of particular interest since IL-1rn is a potent natural inhibitor of proinflammatory IL-1. Using RT-PCR, real-time PCR, immunoblotting and ELISA, we demonstrated that IL-1rn was induced by DCs stimulated with LPS in the absence of TLR4. 2-Aminopurine, a pharmacological PKR inhibitor, completely abrogated LPS-induced expression of IL-1rn in TLR4(neg) DCs, suggesting that LPS-induced TLR4-independent expression of IL-1rn might be mediated by PKR pathways. Considering that IL-1rn is a physiological inhibitor of IL-1, TLR4-independent and PKR-dependent pathways might be crucial in counter-balancing proinflammatory effector functions of DCs resulted from TLR4-dependent activation by LPS.

In vitro synergistic effect of gentamicin with the anti-inflammatory agent diclofenac against Listeria monocytogenes

Aims:  A total of nine Listeria monocytogenes strains (seven serotypes) were studied to ascertain whether the non‐steroidal anti‐inflammatory drug diclofenac (Dc) used in combination with the conventional antilisterial antibiotic gentamicin (Gm) or ampicillin (Am) synergistically augments the efficacy of the antibiotic in vitro.

Isolation and identification of a flavone (quercetin) from Butea frondosa bark

A flavone was isolated from the stem bark of Butea frondosa (Leguminosae). It was given a working name of BF-1 and characterized by m.p., 309–311°C and an empirical formula of C15H10O7. On the basis of chemical and spectral evidence and upon comparison with the literature data, the isolated compound is identified for the first time as quercetin.

Activity of the phenothiazine methdilazine alone or in combination with isoniazid or streptomycin against Mycobacterium tuberculosis in mice.

In previous studies by our group and others, methdilazine (Md), an extensively used antihistaminic phenothiazine, demonstrated broad-spectrum antibacterial activity (Chattopadhyay et al., 1988, 1998; Basu et al., 2005). Md also inhibited various Mycobacterium spp. at 5–15 mg l 21 in vitro and in vivo (Chakrabarty et al., 1993). It has been reported to act by enhancement of streptomycin (Sm) or kanamycin activity against Escherichia coli and Staphylococcus aureus (Chattopadhyay et al., 1988), and by damaging the bacterial cytoplasmic membrane (Chattopadhyay et al., 1998) or, like other phenothiazines, by reversing clinical resistance (Kristiansen et al., 2007) and inhibiting efflux pumps (Amaral et al., 2008).

Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine

Abstract It is believed today that nucleocapsid protein (N) of severe acute respiratory syndrome (SARS)-CoV is one of the most promising antigen candidates for vaccine design. In this study, three fragments [N1 (residues: 1–422); N2 (residues: 1–109); N3 (residues: 110–422)] of N protein of SARS-CoV were expressed in Escherichia coli and analyzed by pooled sera of convalescence phase of SARS patients. Three gene fragments [N1 (1–1269 nt), N2 (1–327 nt) and N3 (328–1269 nt)—expressing the same proteins of N1, N2 and N3, respectively] of SARS-N were cloned into pVAX-1 and used to immunize BALB/c mice by electroporation. Humoral (by enzyme-linked immunosorbent assay, ELISA) and cellular (by cell proliferation and CD4+:CD8+ assay) immunity was detected by using recombinant N1 and N3 specific antigen. Results showed that N1 and N3 fragments of N protein expressed by E. coli were able to react with sera of SARS patients but N2 could not. Specific humoral and cellular immunity in mice could be induced significantly by inoculating SARS-CoV N1 and N3 DNA vaccine. In addition, the immune response levels in N3 were significantly higher for antibody responses (IgG and IgG1 but not IgG2a) and cell proliferation but not in CD4+:CD8+ assay compared to N1 vaccine. The identification of antigenic N protein fragments has implications to provide basic information for the design of DNA vaccine against SARS-CoV. The present results not only suggest that DNA immunization with pVax-N3 could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also illustrates that gene immunization with these SARS DNA vaccines can generate different immune responses.

Experimental analyses of synergistic combinations of antibiotics with a recently recognised antibacterial agent, lacidipine

The cardiovascular drug lacidipine (Lc) is known to possess antibacterial activity. Further potentiation of action is possible by synergism between Lc and an antibiotic or a non-antibiotic. The minimum inhibitory concentration (MIC) of antibiotics, Lc and other non-antibiotics were detected by the agar dilution technique in different bacteria. Synergism was determined by disc diffusion assay, the fractional inhibitory concentration (FIC) index through checkerboard assessment and, also, the protective capacity of the combination by administering the drugs along with 50 × LD50 challenge dose of virulent Salmonella typhimurium in animal experiments. Synergism between Lc and penicillin was found to be statistically significant (P ≤ 0.01) when compared with their individual effects. The FIC index of this combination was 0.375, confirming synergism. In vivo tests suggested the statistically significant protection of infected mice with this combination. Lc exhibited synergism when combined with non-antibiotics methdilazine and triflupromazine both in vitro and in vivo. Distinct antimicrobial action of Lc and its subsequent synergism with other drugs can open up the possibility of synthesising new molecules by the structural analyses of these compounds.