Mohammad Azad - Academia.edu (original) (raw)

Papers by Mohammad Azad

Frontiers in Microbiology

The Journal of antimicrobial chemotherapy, Jan 23, 2018

Current inhaled polymyxin therapy is empirical and often large doses are administered, which can ... more Current inhaled polymyxin therapy is empirical and often large doses are administered, which can lead to pulmonary adverse effects. There is a dearth of information on the mechanisms of polymyxin-induced lung toxicity and their intracellular localization in lung epithelial cells. To investigate the intracellular localization of polymyxins in human lung epithelial A549 cells. A549 cells were treated with polymyxin B and intracellular organelles (early and late endosomes, endoplasmic reticulum, mitochondria, lysosomes and autophagosomes), ubiquitin protein and polymyxin B were visualized using immunostaining and confocal microscopy. Fluorescence intensities of the organelles and polymyxin B were quantified and correlated for co-localization using ImageJ and Imaris platforms. Polymyxin B co-localized with early endosomes, lysosomes and ubiquitin at 24 h. Significantly increased lysosomal activity and the autophagic protein LC3A were observed after 0.5 and 1.0 mM polymyxin B treatment a...

Antimicrobial agents and chemotherapy, Jan 23, 2017

Polymyxins are a last-line defence against multidrug-resistant Gram-negative pathogens. Recent ph... more Polymyxins are a last-line defence against multidrug-resistant Gram-negative pathogens. Recent pharmacological data show that intravenous polymyxins can cause nephrotoxicity in up to 60% of patients, and plasma concentrations of polymyxins are suboptimal in a large proportion of patients with the currently recommended dosage regimens. Simply increasing the daily dose of polymyxins is not possible due to nephrotoxicity. This study aimed to examine the protective effect of methionine against polymyxin-induced nephrotoxicity. Methionine (400 mg/kg), polymyxin B (35 mg/kg), a combination of methionine (100 or 400 mg/kg) and polymyxin B, and saline were administered in mice twice daily over 3.5 days. Kidneys were collected immediately at the end of the experiment for histological examination. Effect of methionine on the pharmacokinetics of polymyxin B was investigated in rats. Attenuation of polymyxin B (0.75 mM) induced mitochondrial superoxide production by methionine (10.0 mM) was exa...

Antimicrobial Agents and Chemotherapy

Colistin therapy is used as the last-line of defence against life-threatening Gram-negative infec... more Colistin therapy is used as the last-line of defence against life-threatening Gram-negative infections. Nephrotoxicity is the major dose-limiting side-effect that impedes optimal dosing of patients. This study aims to examine the nephroprotective effect of the plasma volume expander gelofusine against colistin-induced nephrotoxicity. Renal protection was assessed in mice that were subcutaneously injected with colistin sulphate (14 mg/kg x 6 doses every 2 h; accumulated dose 84 mg/kg) and simultaneously injected in the intraperitoneal region with gelofusine (75, 150, 300 or 600 mg/kg x 6). At 2 and 20 h after the last colsitin dose, mice were euthanized and severity of renal alteration was examined histologically. Histological findings in mice revealed that colistin-induced nephrotoxicity was ameliorated by gelofusine in a dose-dependent manner, whereas significant histological abnormalities were detected in the kidneys of mice in the colistin only group. The impact of co-administere...

Antimicrobial agents and chemotherapy, Jun 17, 2017

Inhaled polymyxins are of considerable utility to achieve optimal exposure in the respiratory tra... more Inhaled polymyxins are of considerable utility to achieve optimal exposure in the respiratory tract for the treatment of lung infections caused by multidrug-resistant Gram-negative pathogens. Current inhaled polymyxin therapy is empirical and often high doses are used which may lead to potential pulmonary adverse effects. This study aimed to investigate the effect of polymyxins on human lung epithelial cells (A549). Viability of A549 cells was examined after treatment with polymyxins using flow cytometry. Activation of caspase-3, -8, -9, expression of Fas ligand (FasL), loss of mitochondrial membrane potential and mitochondrial oxidative stress induced by polymyxin B was evaluated. The EC50 (95% CI) value of polymyxin B induced cell death was 1.74 mM (1.60 to 1.90 mM). Colistin was at least two-fold less toxic than polymyxin B while colistimethate (CMS) was non-toxic. With 2.0 mM polymyxin B, apoptotic cells were 30.6 ± 11.5% (mean ± SD) at 8 h and increased to 71.3 ± 3.72% at 24 h....

ACS infectious diseases, Jul 8, 2016

Novel combination therapies are desperately needed for combating lung infections caused by bacter... more Novel combination therapies are desperately needed for combating lung infections caused by bacterial "superbugs". This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the cystic fibrosis (CF) drugs KALYDECO (ivacaftor) and ORKAMBI (ivacaftor + lumacaftor) against Gram-negative pathogens that commonly colonize the CF lung, in particular, the problematic Pseudomonas aeruginosa. The in vitro synergistic activity of polymyxin B combined with ivacaftor or lumacaftor was assessed using checkerboard and static time-kill assays against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa isolates from the lungs of CF patients. Polymyxin B, ivacaftor, and lumacaftor were ineffective when used individually against polymyxin-resistant (MIC ≥ 4 mg/L) isolates. However, when used together, the combination of clinically relevant concentrations of polymyxin B (2 mg/L) combined with ivacaftor (8 mg/L) or ivacaftor (8 m...

ACS Infectious Diseases, 2015

Polymyxin B and colistin are currently used as a &amp... more Polymyxin B and colistin are currently used as a 'last-line' treatment for multidrug-resistant Gram-negative bacteria. However very little is known about the pharmacological differences between polymyxin B1, polymyxin B2, colistin A, colistin B, the major cyclic lipopeptides components present in polymyxin B and colistin products. Here, we report on the in vitro and in vivo antimicrobial activity and toxicity of these major lipopeptide components. All four lipopeptides had comparable MICs (<0.125-4 mg/L) against a panel of clinical Gram-negative isolates. They also had comparable in vivo antimicrobial activity (Δlog10 CFU/mL >-3) and nephrotoxicity (mild to moderate histological damage) in mouse models. However, polymyxin B1 and colistin A showed significantly higher (> 3-fold) in vitro apoptotic effect on human kidney proximal tubular HK-2 cells than polymyxin B2 and colistin B, respectively. Compared to the commercial polymyxin and colistin products, the individual lipopeptide components had slightly more in vivo antimicrobial activity. Our results highlight the need to re-assess pharmacopoeial standards for polymyxins B and colistin and to standardize the composition of the different commercial products of polymyxin antibiotics.

Antimicrobial agents and chemotherapy, Jan 21, 2015

Polymyxins are cyclic lipopeptide antibiotics that serve as a last-line defence against Gram-nega... more Polymyxins are cyclic lipopeptide antibiotics that serve as a last-line defence against Gram-negative 'superbugs'. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular cells (NRK-52E). Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labelled mono-dansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543 and MIPS-9544) were designed, synthesized and screened for antimicrobial activity and apoptotic effect against rat kidney proximal tubular cells. Based on the assessment of antimicrobial activity, cellular uptake and apoptotic effect on ren...

Antimicrobial agents and chemotherapy, Jan 26, 2015

Identifying the pathways involved in the apoptotic cell death that is associated with polymyxin-i... more Identifying the pathways involved in the apoptotic cell death that is associated with polymyxin-induced nephrotoxicity is crucial for the development of strategies to ameliorate this dose-limiting side-effect and for the development of novel safer polymyxins. The primary aim of this study is to identify the major pathways which lead to polymyxin-induced apoptosis in cultured rat kidney proximal tubular cells (NRK-52E). Caspase-3, 8 and 9 were activated by polymyxin B treatment in a concentration-dependent manner. Concentration- and time-dependent expression of FasL and deformation of mitochondrial morphology were revealed following polymyxin B treatment. The percentage of cells with filamentous mitochondria (regular morphology) following 8-h treatment with 1.0 mM polymyxin B was 56.2 ± 9.7% (n = 3). This was decreased to 30.7 ± 7.5% when the polymyxin B concentration was increased to 2.0 mM polymyxin B. The mitochondrial membrane potential (Δψm) decreased to 14.1 ± 2.9% in the cells...

Pakistan journal of pharmaceutical sciences, 2009

This investigation describes the preparation and in vitro evaluation of gastroretentive floating ... more This investigation describes the preparation and in vitro evaluation of gastroretentive floating tablet of theophylline. Two hydrophilic cellulose derivatives, Methocel K100M and Methocel K15MCR were evaluated for their gel forming and release controlling properties. Sodium bicarbonate and citric acid were incorporated as gas generating agents. The effects of soluble components (sodium bicarbonate and citric acid), gel forming agents and amount variation of theophylline on drug release profile and floating properties were investigated. Tablets were prepared by direct compression technique. Formulations were evaluated for in vitro buoyancy and drug release study was evaluated for eight hours using USP XXII paddle-type dissolution apparatus using 0.1N HCl as dissolution medium. The release mechanisms were explored and explained with zero order, first order, Higuchi and Korsmeyer equations. The release rate, extent and mechanisms were found to be governed by polymer and floating agent ...

Innate immunity, 2013

The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and... more The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and polymyxins B and E was examined with fluorometric and calorimetric methods, and by (1)H NMR, using a paired wild type (WT) and the ΔlpxM mutant strains B5055 and B5055ΔlpxM, which predominantly express LPS with hexa- and penta-acylated lipid A structures respectively. LPS from B5055ΔlpxM displayed a fourfold increased binding affinity for polymyxins B and E compared with the B5055 WT LPS. EC50 values were consistent with polymyxin minimum inhibitory concentration (MIC) values for each strain. Accordingly, polymyxin exposure considerably enhanced the permeability of the B5055ΔlpxM OM. Analysis of the melting profiles of isolated LPS aggregates suggested that bactericidal polymyxin activity may relate to the acyl chains' phase of the outer membrane (OM). The enhanced polymyxin susceptibility of B5055ΔlpxM may be attributable to the favorable insertion of polymyxins into the more fluid...

Antimicrobial agents and chemotherapy, 2014

The Journal of antimicrobial chemotherapy, 2015

Dose-limiting nephrotoxicity remains the Achilles' heel of polymyxin B and polymyxin E (also ... more Dose-limiting nephrotoxicity remains the Achilles' heel of polymyxin B and polymyxin E (also known as colistin), which are important last-line antibiotics used against infections caused by MDR Gram-negative 'superbugs'. An understanding of the mechanisms of nephrotoxicity, including renal tissue distribution, is crucial for the development of safer polymyxin lipopeptide antibiotics. This is the first study to visualize the kidney distribution of polymyxin B using a mouse nephrotoxicity model and in situ immunostaining of kidney sections. Polymyxin B nephrotoxicity in mice was induced over the course of 3 days (accumulated intravenous dose 175 mg/kg) and kidneys were harvested and frozen sectioned. The sections were fixed in cold acetone, dried and treated with 1% hydrogen peroxide. Endogenous mouse immunoglobulins were blocked and the tissue sections were treated with anti-polymyxin B mouse IgM antibody. The sections were incubated with a biotinylated anti-mouse secondar...

Analytical chemistry, Jan 3, 2015

Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limit... more Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limiting nephrotoxicity can occur in up to 60% of patients after intravenous administration. Understanding the accumulation and concentration of polymyxin within renal tubular cells is essential for the development of novel strategies to ameliorate its nephrotoxicity and to develop safer, new polymyxins. We designed and synthesized a novel dual-modality iodine-labeled fluorescent probe for quantitative mapping of polymyxin in kidney proximal tubular cells. Measured by synchrotron X-ray fluorescence microscopy, polymyxin concentrations in single rat (NRK-52E) and human (HK-2) kidney tubular cells were approximately 1930- to 4760-fold higher than extracellular concentrations. Our study is the first to quantitatively measure the significant uptake of polymyxin in renal tubular cells and provides crucial information for the understanding of polymyxin-induced nephrotoxicity. Importantly, our appro...

Innate Immunity, 2014

This study examines the interaction of polymyxin B and colistin with the surface and outer membra... more This study examines the interaction of polymyxin B and colistin with the surface and outer membrane components of a susceptible and resistant strain of Klebsiella pneumoniae. The interaction between polymyxins and bacterial membrane and isolated LPS from paired wild type and polymyxin-resistant strains of K. pneumoniae were examined with N-phenyl-1-naphthylamine (NPN) uptake, fluorometric binding and thermal shift assays, lysozyme and deoxycholate sensitivity assays, and by (1)H NMR. LPS from the polymyxin-resistant strain displayed a reduced binding affinity for polymyxins B and colistin in comparison with the wild type LPS. The outer membrane NPN permeability of the resistant strain was greater compared with the susceptible strain. Polymyxin exposure enhanced the permeability of the outer membrane of the wild type strain to lysozyme and deoxycholate, whereas polymyxin concentrations up to 32 mg/ml failed to permeabilize the outer membrane of the resistant strain. Zeta potential measurements revealed that mid-logarithmic phase wild type cells exhibited a greater negative charge than the mid-logarithmic phase-resistant cells. Taken together, our findings suggest that the resistant derivative of K. pneumoniae can block the electrostatically driven first stage of polymyxin action, which thereby renders the hydrophobically driven second tier of polymyxin action on the outer membrane inconsequential.

Pakistan journal of pharmaceutical sciences, 2009

The purpose of the present study was to investigate the effect of channeling agent on the release... more The purpose of the present study was to investigate the effect of channeling agent on the release profile of theophylline from Kollidon SR based matrix systems. Matrix tablets of theophylline using Kollidon SR which is plastic in nature were prepared by direct compression process. NaCl and PEG 1500 were used as channeling agents. Drug release study was evaluated for eight hours using USP 22 paddle-type dissolution apparatus using distilled water as the dissolution medium. The release mechanisms were explored and explained with zero order, Higuchi, first order and Korsmeyer equations. The release rate, extent and mechanisms were found to be governed by the type and content of the channeling agents. Increased rate and extent of the drug release were found by using higher content of channeling agent (42.49%) in the matrix due to increased porosity when compared with the formulation having no channeling agents. On the other hand decreased rate and extent of drug release were observed in...

Pakistan journal of pharmaceutical sciences, 2010

The objective of this study was to observe the drug interaction between levofloxacin and omeprazo... more The objective of this study was to observe the drug interaction between levofloxacin and omeprazole using urinary excretion data. Levofloxacin tablet and omeprazole capsule were administered separately as well as in combination in fasting condition with a wash out period of two weeks after each administration. Urine was collected at different time intervals of 0, 0-2, 2-4, 4-8, 8-12, 12-24, 24-36 and 36-48 hr post-dose and analyzed using a validated HPLC with UV detection. Different pharmacokinetic parameters for both drugs were determined using non-compartmental method. The maximum rate of excretion (R(max)) of levofloxacin was not decreased significantly when co-administered with omeprazole (p<0.05). Similarly no significant difference (p = 0.350) was observed for Rmax of omeprazole when co-administered with levofloxacin. Again the fraction of levofloxacin excreted (f(e)/f) was not changed significantly (p = 0.953) due to the co-administration of omeprazole. Similarly fraction ...

Dhaka University Journal of Pharmaceutical Sciences, 2011

The objective of this study was to develop a simple, efficient, precise and accurate reverse-phas... more The objective of this study was to develop a simple, efficient, precise and accurate reverse-phase HPLC method for the simultaneous determination of metformin in combination with gliclazide in newly formulated tablets. Chromatographic determination was performed on a reversed phase C18 column (2.6 mm x 250 mm; 5 μm particle size) using a mixture of buffer (1 ml of orthophosphoric acid with 1 ml triethylamine upto 1000 ml with HPLC grade water) and methanol at the ratio of 60:40 as mobile phase at a flow rate of 1ml/min. The UV detection was set at 230 nm. Under the developed conditions, good separation of the analytes was achieved. The calibration curves were linear with coefficient correlation between 0.998 to 1.0 for both drugs over a concentration range of 1 to 50 μg/ml for metformin hydrochloride and 0.16 to 8 μg/ml for gliclazide. The method was also validated in terms of precision (RSD = 0.06 to 3.22%) and accuracy (percent deviation = 0.049 to 2.602%). The proposed method was...

Dhaka University Journal of Pharmaceutical Sciences, 2007

The purpose of the study was to develop a simple, sensitive and rapid RP-HPLC method for the dete... more The purpose of the study was to develop a simple, sensitive and rapid RP-HPLC method for the determination of desloratadine in marketed products. Chromatographic determination was performed in a reverse phase C18 column (250 mm × 3.3 mm I.D. , 5?m particle size) using a mixture of acetonitrile ? n-pentane sulphonic acid sodium salt monohydrate, adjusted to pH 3.0± 0.05 with phosphoric acid (60? 40 v/v) as mobile phase and delivered at a flow rate of 1 ml/min. The UV detection was set at 254 nm. The calibration range was from 2.0 to 40 ?g/ml. The method was validated in term of linearity (r2>0.98, RSD= 1.958%), precision (RSD=3.757 %) and accuracy (deviation>2.653%, RSD> 2.203%). The limit of quantification was 2 ?g/ml and the limit of detection was 0.1 ?g/ml. The linear ranges of desloratadine were 20.23 ± 0.368 ?g/ml and 6.545 ± 0.0495 ?g/ml in tablet (potency = 99.175 ± 0.718 %) and syrup (potency = 101.15 ± 1.838 %) respectively. The potency of desloratadine in marketed ...

Frontiers in Microbiology

The Journal of antimicrobial chemotherapy, Jan 23, 2018

Current inhaled polymyxin therapy is empirical and often large doses are administered, which can ... more Current inhaled polymyxin therapy is empirical and often large doses are administered, which can lead to pulmonary adverse effects. There is a dearth of information on the mechanisms of polymyxin-induced lung toxicity and their intracellular localization in lung epithelial cells. To investigate the intracellular localization of polymyxins in human lung epithelial A549 cells. A549 cells were treated with polymyxin B and intracellular organelles (early and late endosomes, endoplasmic reticulum, mitochondria, lysosomes and autophagosomes), ubiquitin protein and polymyxin B were visualized using immunostaining and confocal microscopy. Fluorescence intensities of the organelles and polymyxin B were quantified and correlated for co-localization using ImageJ and Imaris platforms. Polymyxin B co-localized with early endosomes, lysosomes and ubiquitin at 24 h. Significantly increased lysosomal activity and the autophagic protein LC3A were observed after 0.5 and 1.0 mM polymyxin B treatment a...

Antimicrobial agents and chemotherapy, Jan 23, 2017

Polymyxins are a last-line defence against multidrug-resistant Gram-negative pathogens. Recent ph... more Polymyxins are a last-line defence against multidrug-resistant Gram-negative pathogens. Recent pharmacological data show that intravenous polymyxins can cause nephrotoxicity in up to 60% of patients, and plasma concentrations of polymyxins are suboptimal in a large proportion of patients with the currently recommended dosage regimens. Simply increasing the daily dose of polymyxins is not possible due to nephrotoxicity. This study aimed to examine the protective effect of methionine against polymyxin-induced nephrotoxicity. Methionine (400 mg/kg), polymyxin B (35 mg/kg), a combination of methionine (100 or 400 mg/kg) and polymyxin B, and saline were administered in mice twice daily over 3.5 days. Kidneys were collected immediately at the end of the experiment for histological examination. Effect of methionine on the pharmacokinetics of polymyxin B was investigated in rats. Attenuation of polymyxin B (0.75 mM) induced mitochondrial superoxide production by methionine (10.0 mM) was exa...

Antimicrobial Agents and Chemotherapy

Colistin therapy is used as the last-line of defence against life-threatening Gram-negative infec... more Colistin therapy is used as the last-line of defence against life-threatening Gram-negative infections. Nephrotoxicity is the major dose-limiting side-effect that impedes optimal dosing of patients. This study aims to examine the nephroprotective effect of the plasma volume expander gelofusine against colistin-induced nephrotoxicity. Renal protection was assessed in mice that were subcutaneously injected with colistin sulphate (14 mg/kg x 6 doses every 2 h; accumulated dose 84 mg/kg) and simultaneously injected in the intraperitoneal region with gelofusine (75, 150, 300 or 600 mg/kg x 6). At 2 and 20 h after the last colsitin dose, mice were euthanized and severity of renal alteration was examined histologically. Histological findings in mice revealed that colistin-induced nephrotoxicity was ameliorated by gelofusine in a dose-dependent manner, whereas significant histological abnormalities were detected in the kidneys of mice in the colistin only group. The impact of co-administere...

Antimicrobial agents and chemotherapy, Jun 17, 2017

Inhaled polymyxins are of considerable utility to achieve optimal exposure in the respiratory tra... more Inhaled polymyxins are of considerable utility to achieve optimal exposure in the respiratory tract for the treatment of lung infections caused by multidrug-resistant Gram-negative pathogens. Current inhaled polymyxin therapy is empirical and often high doses are used which may lead to potential pulmonary adverse effects. This study aimed to investigate the effect of polymyxins on human lung epithelial cells (A549). Viability of A549 cells was examined after treatment with polymyxins using flow cytometry. Activation of caspase-3, -8, -9, expression of Fas ligand (FasL), loss of mitochondrial membrane potential and mitochondrial oxidative stress induced by polymyxin B was evaluated. The EC50 (95% CI) value of polymyxin B induced cell death was 1.74 mM (1.60 to 1.90 mM). Colistin was at least two-fold less toxic than polymyxin B while colistimethate (CMS) was non-toxic. With 2.0 mM polymyxin B, apoptotic cells were 30.6 ± 11.5% (mean ± SD) at 8 h and increased to 71.3 ± 3.72% at 24 h....

ACS infectious diseases, Jul 8, 2016

Novel combination therapies are desperately needed for combating lung infections caused by bacter... more Novel combination therapies are desperately needed for combating lung infections caused by bacterial "superbugs". This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the cystic fibrosis (CF) drugs KALYDECO (ivacaftor) and ORKAMBI (ivacaftor + lumacaftor) against Gram-negative pathogens that commonly colonize the CF lung, in particular, the problematic Pseudomonas aeruginosa. The in vitro synergistic activity of polymyxin B combined with ivacaftor or lumacaftor was assessed using checkerboard and static time-kill assays against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa isolates from the lungs of CF patients. Polymyxin B, ivacaftor, and lumacaftor were ineffective when used individually against polymyxin-resistant (MIC ≥ 4 mg/L) isolates. However, when used together, the combination of clinically relevant concentrations of polymyxin B (2 mg/L) combined with ivacaftor (8 mg/L) or ivacaftor (8 m...

ACS Infectious Diseases, 2015

Polymyxin B and colistin are currently used as a &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp... more Polymyxin B and colistin are currently used as a &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;last-line&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; treatment for multidrug-resistant Gram-negative bacteria. However very little is known about the pharmacological differences between polymyxin B1, polymyxin B2, colistin A, colistin B, the major cyclic lipopeptides components present in polymyxin B and colistin products. Here, we report on the in vitro and in vivo antimicrobial activity and toxicity of these major lipopeptide components. All four lipopeptides had comparable MICs (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.125-4 mg/L) against a panel of clinical Gram-negative isolates. They also had comparable in vivo antimicrobial activity (Δlog10 CFU/mL &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;-3) and nephrotoxicity (mild to moderate histological damage) in mouse models. However, polymyxin B1 and colistin A showed significantly higher (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 3-fold) in vitro apoptotic effect on human kidney proximal tubular HK-2 cells than polymyxin B2 and colistin B, respectively. Compared to the commercial polymyxin and colistin products, the individual lipopeptide components had slightly more in vivo antimicrobial activity. Our results highlight the need to re-assess pharmacopoeial standards for polymyxins B and colistin and to standardize the composition of the different commercial products of polymyxin antibiotics.

Antimicrobial agents and chemotherapy, Jan 21, 2015

Polymyxins are cyclic lipopeptide antibiotics that serve as a last-line defence against Gram-nega... more Polymyxins are cyclic lipopeptide antibiotics that serve as a last-line defence against Gram-negative 'superbugs'. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular cells (NRK-52E). Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labelled mono-dansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543 and MIPS-9544) were designed, synthesized and screened for antimicrobial activity and apoptotic effect against rat kidney proximal tubular cells. Based on the assessment of antimicrobial activity, cellular uptake and apoptotic effect on ren...

Antimicrobial agents and chemotherapy, Jan 26, 2015

Identifying the pathways involved in the apoptotic cell death that is associated with polymyxin-i... more Identifying the pathways involved in the apoptotic cell death that is associated with polymyxin-induced nephrotoxicity is crucial for the development of strategies to ameliorate this dose-limiting side-effect and for the development of novel safer polymyxins. The primary aim of this study is to identify the major pathways which lead to polymyxin-induced apoptosis in cultured rat kidney proximal tubular cells (NRK-52E). Caspase-3, 8 and 9 were activated by polymyxin B treatment in a concentration-dependent manner. Concentration- and time-dependent expression of FasL and deformation of mitochondrial morphology were revealed following polymyxin B treatment. The percentage of cells with filamentous mitochondria (regular morphology) following 8-h treatment with 1.0 mM polymyxin B was 56.2 ± 9.7% (n = 3). This was decreased to 30.7 ± 7.5% when the polymyxin B concentration was increased to 2.0 mM polymyxin B. The mitochondrial membrane potential (Δψm) decreased to 14.1 ± 2.9% in the cells...

Pakistan journal of pharmaceutical sciences, 2009

This investigation describes the preparation and in vitro evaluation of gastroretentive floating ... more This investigation describes the preparation and in vitro evaluation of gastroretentive floating tablet of theophylline. Two hydrophilic cellulose derivatives, Methocel K100M and Methocel K15MCR were evaluated for their gel forming and release controlling properties. Sodium bicarbonate and citric acid were incorporated as gas generating agents. The effects of soluble components (sodium bicarbonate and citric acid), gel forming agents and amount variation of theophylline on drug release profile and floating properties were investigated. Tablets were prepared by direct compression technique. Formulations were evaluated for in vitro buoyancy and drug release study was evaluated for eight hours using USP XXII paddle-type dissolution apparatus using 0.1N HCl as dissolution medium. The release mechanisms were explored and explained with zero order, first order, Higuchi and Korsmeyer equations. The release rate, extent and mechanisms were found to be governed by polymer and floating agent ...

Innate immunity, 2013

The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and... more The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and polymyxins B and E was examined with fluorometric and calorimetric methods, and by (1)H NMR, using a paired wild type (WT) and the ΔlpxM mutant strains B5055 and B5055ΔlpxM, which predominantly express LPS with hexa- and penta-acylated lipid A structures respectively. LPS from B5055ΔlpxM displayed a fourfold increased binding affinity for polymyxins B and E compared with the B5055 WT LPS. EC50 values were consistent with polymyxin minimum inhibitory concentration (MIC) values for each strain. Accordingly, polymyxin exposure considerably enhanced the permeability of the B5055ΔlpxM OM. Analysis of the melting profiles of isolated LPS aggregates suggested that bactericidal polymyxin activity may relate to the acyl chains' phase of the outer membrane (OM). The enhanced polymyxin susceptibility of B5055ΔlpxM may be attributable to the favorable insertion of polymyxins into the more fluid...

Antimicrobial agents and chemotherapy, 2014

The Journal of antimicrobial chemotherapy, 2015

Dose-limiting nephrotoxicity remains the Achilles' heel of polymyxin B and polymyxin E (also ... more Dose-limiting nephrotoxicity remains the Achilles' heel of polymyxin B and polymyxin E (also known as colistin), which are important last-line antibiotics used against infections caused by MDR Gram-negative 'superbugs'. An understanding of the mechanisms of nephrotoxicity, including renal tissue distribution, is crucial for the development of safer polymyxin lipopeptide antibiotics. This is the first study to visualize the kidney distribution of polymyxin B using a mouse nephrotoxicity model and in situ immunostaining of kidney sections. Polymyxin B nephrotoxicity in mice was induced over the course of 3 days (accumulated intravenous dose 175 mg/kg) and kidneys were harvested and frozen sectioned. The sections were fixed in cold acetone, dried and treated with 1% hydrogen peroxide. Endogenous mouse immunoglobulins were blocked and the tissue sections were treated with anti-polymyxin B mouse IgM antibody. The sections were incubated with a biotinylated anti-mouse secondar...

Analytical chemistry, Jan 3, 2015

Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limit... more Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limiting nephrotoxicity can occur in up to 60% of patients after intravenous administration. Understanding the accumulation and concentration of polymyxin within renal tubular cells is essential for the development of novel strategies to ameliorate its nephrotoxicity and to develop safer, new polymyxins. We designed and synthesized a novel dual-modality iodine-labeled fluorescent probe for quantitative mapping of polymyxin in kidney proximal tubular cells. Measured by synchrotron X-ray fluorescence microscopy, polymyxin concentrations in single rat (NRK-52E) and human (HK-2) kidney tubular cells were approximately 1930- to 4760-fold higher than extracellular concentrations. Our study is the first to quantitatively measure the significant uptake of polymyxin in renal tubular cells and provides crucial information for the understanding of polymyxin-induced nephrotoxicity. Importantly, our appro...

Innate Immunity, 2014

This study examines the interaction of polymyxin B and colistin with the surface and outer membra... more This study examines the interaction of polymyxin B and colistin with the surface and outer membrane components of a susceptible and resistant strain of Klebsiella pneumoniae. The interaction between polymyxins and bacterial membrane and isolated LPS from paired wild type and polymyxin-resistant strains of K. pneumoniae were examined with N-phenyl-1-naphthylamine (NPN) uptake, fluorometric binding and thermal shift assays, lysozyme and deoxycholate sensitivity assays, and by (1)H NMR. LPS from the polymyxin-resistant strain displayed a reduced binding affinity for polymyxins B and colistin in comparison with the wild type LPS. The outer membrane NPN permeability of the resistant strain was greater compared with the susceptible strain. Polymyxin exposure enhanced the permeability of the outer membrane of the wild type strain to lysozyme and deoxycholate, whereas polymyxin concentrations up to 32 mg/ml failed to permeabilize the outer membrane of the resistant strain. Zeta potential measurements revealed that mid-logarithmic phase wild type cells exhibited a greater negative charge than the mid-logarithmic phase-resistant cells. Taken together, our findings suggest that the resistant derivative of K. pneumoniae can block the electrostatically driven first stage of polymyxin action, which thereby renders the hydrophobically driven second tier of polymyxin action on the outer membrane inconsequential.

Pakistan journal of pharmaceutical sciences, 2009

The purpose of the present study was to investigate the effect of channeling agent on the release... more The purpose of the present study was to investigate the effect of channeling agent on the release profile of theophylline from Kollidon SR based matrix systems. Matrix tablets of theophylline using Kollidon SR which is plastic in nature were prepared by direct compression process. NaCl and PEG 1500 were used as channeling agents. Drug release study was evaluated for eight hours using USP 22 paddle-type dissolution apparatus using distilled water as the dissolution medium. The release mechanisms were explored and explained with zero order, Higuchi, first order and Korsmeyer equations. The release rate, extent and mechanisms were found to be governed by the type and content of the channeling agents. Increased rate and extent of the drug release were found by using higher content of channeling agent (42.49%) in the matrix due to increased porosity when compared with the formulation having no channeling agents. On the other hand decreased rate and extent of drug release were observed in...

Pakistan journal of pharmaceutical sciences, 2010

The objective of this study was to observe the drug interaction between levofloxacin and omeprazo... more The objective of this study was to observe the drug interaction between levofloxacin and omeprazole using urinary excretion data. Levofloxacin tablet and omeprazole capsule were administered separately as well as in combination in fasting condition with a wash out period of two weeks after each administration. Urine was collected at different time intervals of 0, 0-2, 2-4, 4-8, 8-12, 12-24, 24-36 and 36-48 hr post-dose and analyzed using a validated HPLC with UV detection. Different pharmacokinetic parameters for both drugs were determined using non-compartmental method. The maximum rate of excretion (R(max)) of levofloxacin was not decreased significantly when co-administered with omeprazole (p<0.05). Similarly no significant difference (p = 0.350) was observed for Rmax of omeprazole when co-administered with levofloxacin. Again the fraction of levofloxacin excreted (f(e)/f) was not changed significantly (p = 0.953) due to the co-administration of omeprazole. Similarly fraction ...

Dhaka University Journal of Pharmaceutical Sciences, 2011

The objective of this study was to develop a simple, efficient, precise and accurate reverse-phas... more The objective of this study was to develop a simple, efficient, precise and accurate reverse-phase HPLC method for the simultaneous determination of metformin in combination with gliclazide in newly formulated tablets. Chromatographic determination was performed on a reversed phase C18 column (2.6 mm x 250 mm; 5 μm particle size) using a mixture of buffer (1 ml of orthophosphoric acid with 1 ml triethylamine upto 1000 ml with HPLC grade water) and methanol at the ratio of 60:40 as mobile phase at a flow rate of 1ml/min. The UV detection was set at 230 nm. Under the developed conditions, good separation of the analytes was achieved. The calibration curves were linear with coefficient correlation between 0.998 to 1.0 for both drugs over a concentration range of 1 to 50 μg/ml for metformin hydrochloride and 0.16 to 8 μg/ml for gliclazide. The method was also validated in terms of precision (RSD = 0.06 to 3.22%) and accuracy (percent deviation = 0.049 to 2.602%). The proposed method was...

Dhaka University Journal of Pharmaceutical Sciences, 2007

The purpose of the study was to develop a simple, sensitive and rapid RP-HPLC method for the dete... more The purpose of the study was to develop a simple, sensitive and rapid RP-HPLC method for the determination of desloratadine in marketed products. Chromatographic determination was performed in a reverse phase C18 column (250 mm × 3.3 mm I.D. , 5?m particle size) using a mixture of acetonitrile ? n-pentane sulphonic acid sodium salt monohydrate, adjusted to pH 3.0± 0.05 with phosphoric acid (60? 40 v/v) as mobile phase and delivered at a flow rate of 1 ml/min. The UV detection was set at 254 nm. The calibration range was from 2.0 to 40 ?g/ml. The method was validated in term of linearity (r2>0.98, RSD= 1.958%), precision (RSD=3.757 %) and accuracy (deviation>2.653%, RSD> 2.203%). The limit of quantification was 2 ?g/ml and the limit of detection was 0.1 ?g/ml. The linear ranges of desloratadine were 20.23 ± 0.368 ?g/ml and 6.545 ± 0.0495 ?g/ml in tablet (potency = 99.175 ± 0.718 %) and syrup (potency = 101.15 ± 1.838 %) respectively. The potency of desloratadine in marketed ...