Potent antitumor activity of a novel cationic pyridinium-ceramide alone or in combination with gemcitabine against human head and neck squamous cell carcinomas in vitro and in vivo

In this study, a cationic water-soluble ceramide analog L-threo-C6-pyridinium-ceramide-bromide (L-t-C6-Pyr-Cer), which exhibits high solubility and bioavailability, inhibited the growth of various human head and neck squamous cell carcinoma (HNSCC) cell lines at low IC50 concentrations, independent of their p53 status. Consistent with its design to target negatively charged intracellular compartments, L-t-C6-Pyr-Cer accumulated mainly in mitochondria-, and nuclei-enriched fractions upon treatment of human UM-SCC-22A cells [human squamous cell carcinoma (SCC) of the hypopharynx] at 1 to 6 h. In addition to its growth-inhibitory function as a single agent, the supra-additive interaction of L-t-C6-Pyr-Cer with gemcitabine (GMZ), a chemotherapeutic agent used in HNSCC, was determined using isobologram studies. Then, the effects of this ceramide, alone or in combination with GMZ, on the growth of UM-SCC-22A xenografts in SCID mice was assessed following the determination of preclinical parameters, such as maximum tolerated dose, clearance from the blood, and bioaccumulation. Results demonstrated that treatment with L-t-C6-Pyr-Cer in combination with GMZ significantly prevented the growth of HNSCC tumors in vivo. The therapeutic efficacy of L-t-C6-Pyr-Cer/GMZ combination against HNSCC tumors was approximately 2.5-fold better than that of the combination of 5-fluorouracil/cis-platin. In addition, liquid chromatography/mass spectroscopy analysis showed that the levels of L-t-C6-Pyr-Cer in HNSCC tumors were significantly higher than its levels in the liver and intestines; interestingly, the combination with GMZ increased the sustained accumulation of this ceramide by approximately 40%. Moreover, treatment with L-t-C6-Pyr-Cer/GMZ combination resulted in a significant inhibition of telomerase activity and decrease in telomere length in vivo, which are among downstream targets of ceramide.     

Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs

Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting.     

Classification of test agent-specific effects in the Syrian hamster embryo assay (pH 6.7) using infrared spectroscopy with computational analysis

The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has utility in the assessment of potential chemical carcinogenicity (both genotoxic and non-genotoxic mechanisms of action). The assay uses morphological transformation as an end point and has a reported sensitivity of 87%, specificity of 83% and overall concordance of 85% with in vivo rodent bioassay data. However, the scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with benzo[a]pyrene, 3-methylcholanthrene, anthracene, N-nitroso-N-methylnitroguanidine, ortho-toluidine HCl, 2,4-diaminotoluene or D-mannitol for 7 days before fixation with methanol. Identified colonies were interrogated by acquiring a minimum of five infrared (IR) spectra per colony using attenuated total reflection Fourier-transform IR spectroscopy. Individual IR spectra were acquired over a spatial area of approximately 250 × 250 μm. Resultant data were analysed using Fisher's linear discriminant analysis and feature histogram algorithms to extract classifying biomarkers of test agent-specific effects or transformation in SHE cells. Clustering of spectral points suggested co-segregation or discrimination of test agent categories based on mechanism of action. Towards transformation, unifying alterations were associated with alterations in the Amide I and Amide II peaks; these were consistently major classifying biomarkers for transformed versus non-transformed SHE cells. Our approach highlights a novel method towards objectively screening and classifying SHE cells, be it to ascertain test agent treatment based on mechanism of action or transformation.     

Evidence of a Major Reservoir of Non-Malarial Febrile Diseases in Malaria-Endemic Regions of Bangladesh

In malaria-endemic regions any febrile case is likely to be classified as malaria based on presumptive diagnosis largely caused by a lack of diagnostic resources. A district-wide prevalence study assessing etiologies of fever in 659 patients recruited in rural and semi-urban areas of Bandarban district in southeastern Bangladesh revealed high proportions of seropositivity for selected infectious diseases (leptospirosis, typhoid fever) potentially being misdiagnosed as malaria because of similarities in the clinical presentation. In an area with point prevalences of more than 40% for malaria among fever cases, even higher seroprevalence rates of leptospirosis and typhoid fever provide evidence of a major persistent reservoir of these pathogens.     

Therapeutic Effectiveness in the Treatment of Experimental Bacterial Keratitis with Ion-activated Mucoadhesive Hydrogel

Purpose: To investigate the therapeutic effectiveness of ion-activated mucoadhesive hydrogel system in the treatment of experimental bacterial keratitis.

Materials and Methods: Mucoadhesive systems were prepared using gellan or sodium alginate alone and combined with sodium carboxymethylcellulose (NaCMC) to enhance the gel bioadhesion properties. The in vivo antimicrobial efficacy of selected mucoadhesive systems was studied in an experiment on bacterial keratitis in rabbit’s eyes and compared with that of the marketed conventional eyedrops.

Results: Ocular tolerance was studied in the eye of albino rabbits and tested formulations were non-irritant with no sign of inflammation. Better improvement in experimental bacterial keratitis in rabbit eyes was observed in animals treated with mucoadhesive hydrogel formulation (GG5 and GS5) compared with marketed drug solution.

Conclusion: The developed system is a viable alternative to conventional eyedrops of GTN due to its ability to enhance bioavailability through its longer precorneal residence time.     

The impact of severe acidemia on neurologic outcome of cardiac arrest survivors undergoing therapeutic hypothermia

Introduction

Therapeutic Hypothermia (TH) has become a standard of care in improving neurological outcomes in cardiac arrest (CA) survivors. Previous studies have defined severe acidemia as plasma pH < 7.20. We investigated the influence of severe acidemia at the time of initiation of TH on neurological outcome in CA survivors.

Methods

A retrospective analysis was performed on 196 consecutive CA survivors (out-of-hospital CA and in-hospital CA) who underwent TH with endovascular cooling between January 2007 and October 2012. Arterial blood gas drawn prior to initiation of TH was utilized to measure pH in all patients. Shockable and non-shockable CA patients were divided into two sub-groups based on pH (pH < 7.2 and pH ≥ 7.2). The primary end-point was measured using the Pittsburgh Cerebral Performance Category (CPC) scale prior to discharge from the hospital: good (CPC 1 and 2) and poor (CPC 3 to 5) neurologic outcome.

Results

Sixty-two percent of shockable CA patients with pH ≥ 7.20 had good neurological outcome as compared to 34% patients with pH < 7.20. Shockable CA patients with pH ≥ 7.20 were 3.3 times more likely to have better neurological outcome when compared to those with pH <7.20 [p = 0.013, OR 3.3, 95% CI (1.28–8.45)]. In comparison, non-shockable CA patients with p ≥ 7.20 did not have a significantly different neurological outcome as compared to those with pH < 7.20 [p = 0.97, OR 1.02, 95% CI (0.31–3.3)].

Conclusion

Presence of severe acidemia at initiation of TH in shockable CA survivors is significantly associated with poor neurological outcomes. This effect was not observed in the non-shockable CA survivors.     

2-Amino-5-thiazolyl motif: a novel scaffold for designing anti-inflammatory agents of diverse structures

Substituted thiazoles with different structural features were synthesized and screened for their anti-inflammatory activity in acute carrageenin induced rat paw edema model and chronic formalin induced rat paw edema model. The compounds 1-5 showed 83, 30, 63, 69 and 73% protection, respectively, in acute carrageenin induced rat paw edema model. In 5-day chronic formalin induced rat paw edema model on the fifth day 1 and 5 gave 66 and 41% protection. Both studies were carried out at a dose of 100mg/kg body weight. The activity was compared with that of Ibuprofen, Rofecoxib, and Dexamethasone both in acute and chronic anti-inflammatory models. Compound 1 without COX-1 and COX-2 inhibitory activity showed good activity profile almost mimicking the gold standard Dexamethasone in terms of efficacy. A 7-day study in rats at dose of 100mg/kg showed that this compound does not have any ulcerogenic activity and toxicity. The activity of 5 shows that incorporating two pharmacophoric features in one molecule can be a good drug designing strategy. 2,4-Diaminothiazoles with an aliphatic oxime esters attached via a ketone bridge to the 5th position of thiazole was identified as a novel scaffold for designing anti-inflammatory agents.