This study aimed to develop a highly sensitive, rapid method to evaluate the pharmacokinetic bioequivalence of two formulations of the anticancer drug, bicalutamide. According to US and Korean regulatory requirements for a bioequivalence test, we developed an achiral, bioanalytical method to determine bicalutamide levels in human plasma. The method included liquid chromatography tandem mass spectrometry in negative mode and was validated with nilutamide as an internal standard. Quantitation was performed for the transition of 429.2 → 255.0 (m/z) for bicalutamide and 316.2 → 273.2 (m/z) for nilutamide. The lower limit of quantitation was 10 ng mL‑1 with a 50 μL plasma sample. This sensitivity was about 8 times higher than current methods in pharmaceuticals and 10 times higher than methods for human plasma samples. The concentrations of seven working standards showed linearity between 10 and 2000 ng mL-1 (r2 ≥ 0.9993). Chromatographic separation was achieved within 4 min, compared to the 10 min of current methods. We used a Luna C18 column (100 mm x 2 mm, 5 μm) with distilled water/acetonitrile (30/70, v/v) as an isocratic mobile phase with a flow rate of 0.3 mL min-1. The average extraction recoveries of 3 quality control concentrations were 94.43% for bicalutamide and 99.28% for nilutamide. The coefficient of variation was ≤15% for intra- and inter-batch assays. Thus, this method satisfied the US and Korean validation requirements. When applied to a pharmacokinetic bioequivalence study in 33 healthy Korean male volunteers, this method showed high sensitivity, selectivity, and accuracy.

Posaconazole and voriconazole, two triazole antifungal agents, are used for the prophylaxis and treatment of invasive mycoses in patients with acute myeloid leukaemia and/or immunocompromised. Inter- and intra-patient variability of pharmacokinetics, drug-drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of both drugs. Therefore, a rapid, selective and sensitive isocratic reversed-phase HPLC assay coupled with Mass spectrometry detection for quantification of posaconazole and voriconazole in serum samples has been developed. Analytes were extracted on solid-phase cartridges (SPE) and chromatographic separation was achieved on a C8 column and detected by mass spectrometry in positive ion mode with the select ion monitoring (SIM) mode. The total chromatographic running time was 6 minutes. The method was successfully used for a pharmacokinetic study but, thanks to its rapidity and selectivity, it’s also suitable for routinarly therapeutic drug monitoring (TDM).

The pharmacokinetic studies of enrofloxacin was conducted in eighteen turkeys (1 to 1.5 years age) weighing between 3 to 4 kg following single i.v. dose (10 mg/kg b.w.) alone and with meloxicam (1 mg/kg b.w.). Quantitative estimation of enrofloxacin and meloxicam was done by high performance liquid chromatography. The maximum concentration of drug in plasma (Cpmax) of enrofloxacin with meloxicam (8.42 ± 0.40 µg/ml) was not significantly different from that of enrofloxacin alone (9.68 ± 0.44 µg/ml). Pharmacokinetic parameters of enrofloxacin alone (C°p =10.38±0.43 µg/ml, t˝β =2.73±0.12 h, MRT = 3.65±0.21 h, ClB =8.41±0.66 ml/kg/min, Vdarea =1.95±0.08 L/kg) as compared to when it was administered with meloxicam ( C°p =9.35±0.62 µg/ml, t˝β =2.70±0.13 h, MRT =3.73±0.18 h, ClB =9.79±0.84 ml/kg/min, Vdarea =2.26±0.15 L/kg) in turkeys did not differ significantly.

Analytical methods for pharmacokinetic studies with quantification of the highly potent anaesthetic drug fentanyl in serum must be rugged, sensitive, precise and accurate. To address these analytical demands and facilitate an automated approach to sample clean-up, a new method was developed for human serum using back-flush column-switching and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). Prior to injection, protein precipitation in trichloroacetic acid was performed in each sample. Samples were diluted with internal standard (D5-fentanyl) in 10% (w/v) trichloroacetic acid, kept at 4oC for 20 min and centrifuged followed by injection of 40µL supernatant onto a BioTrap 500 MS extraction column. Using a time programmed six-port valve switch, the extracted drug was back-flushed onto a Zorbax SB-Aq analytical column, gradient eluted and finally detected after electrospray ionisation with multiple reaction monitoring (MRM) of the transitions m/z 337 → m/z 188 and m/z 342 → m/z 188 for fentanyl and D5-fentanyl, respectively. Mean inter-assay accuracy (n=5) ranged from 93 to 101 % and inter-assay precision(n=5) ranged from 3.7 to 9.4% (C.V.). The method was used in a preliminary study of 4 patients after application of transdermal patches and found fully applicable for future pharmacokinetic studies. This is the first on-line, column-switching LC-MS/MS method validated and demonstrated suitable for quantification of fentanyl in human serum.

The stress dependent tumour cells found to harbour Hsp90 and its inducible component Hsp90a in activated superchaperone complexes are highly sensitive to pharmacological Hsp90 inhibitors compared to normal cells where Hsp90 is present in an uncomplexed state. Downregulating Hsp90a can be achieved using chemical inhibitors and RNAi such as siRNA’s or shRNA’s. This study aimed at assessing the efficacy of siRNA (targeting Hsp90a exon 5) and shRNA (targeting Hsp90a exon 4 and 5 border) in three glioma cell lines (1321N1, GOS-3 and U87- MG). Hsp90a expression at mRNA and protein levels was monitored using qRT-PCR and immunofluorescence, respectively. The downstream effect of silencing Hsp90a was determined by measuring the Akt/PKB kinase activity level. While siRNA treatment decreased Hsp90α mRNA copy numbers by ~35%, shRNA decreased it by ~63% (three glioma cell lines). Furthermore, Hsp90α inhibition by siRNA resulted in downregulating Akt/PKB kinase by ~29%, whereas shRNA downregulated it to ~3% (three glioma cell lines). Considering the vital role of Akt kinase in cell signalling, anti-apoptotic and drug resistant pathways in tumours, the treatment induced sensitivity of Akt to degradation results in a novel therapeutic strategy emphasising a greater potential of shRNA as opposed to siRNA in silencing Hsp90a and subsequently Akt in glioma cells.

This review aims to focus on several of the advantages that an improved understanding of the biology of cancer may offer medicine. Following in the wake of my recent review demonstrating the close molecular mechanistic relationship between heart survival mechanisms and those of cancer under environmental challenge I examine how cancer can provide survival answers for tissues under physiological challenge. I continue the basis of this discussion with the argument that cancer can offer clues as to how to better develop reliable and sound strategies for tissue/organ regeneration. For a number of years the concept with the use of stem cells to enable tissue and/ or organ regeneration has gained attention. Indeed, recently, tooth buds have been regenerated from mouse stem cells allowing complete tooth regeneration in vivo. In this review, the focus is on alternative means for tissue/ organ regeneration by harnessing the all-sort-after property of cellular immortality that cancer possesses in its armamentarium. Literature is presented to demonstrate the utility and practicality of immortal genetic modification of autologous post-mitotic and senescent cells for reimplantation. This process may be of practical value in numerous medical situations from degenerative neurological diseases, wound healing, hair follicle replacement and auditory repair to aiding functional repair of cardiac disease to name but a few. The rational benefits of this technique are presented over stem cell technologies and potential technical obstacles are outlined that may need to be overcome. Novel technologies derived from the use of shuttle expression vectors are outlined here to screen for therapyresistance genes in cancer. These methods may be further enhanced and developed to allow delineation of genes involved in preventing cellular senescence whilst not transforming cells to a cancerous phenotype. These genes can subsequently be used in genetic modification of autologously harvested cells for re-implantation to enable tissue and/or organ regeneration.

Cytomegalovirus (CMV) is one of the most prevalent infectious pathogen in transplant recipients, including those receiving bone marrow or stem cell grafts. Rapid diagnostic tests to identify active CMV infection and preemptive treatment are significant improvements in the management of CMV. Two newly identified beta herpesviruses, human herpesvirus-6 (HHV-6) and human betaherpesvirus-7 (HHV-7), are genetically more closely related to each other than to CMV and have been frequently detected in the blood of allogeneic HSCT. HHV-6 reactivation has been associated with fever, rash, delayed engraftment and encephalitis. Also, HHV-7 has been reported as a cause of severe central nervous system disease and with severe GVHD and sepsis secondary to immune suppression. Nested polymerase chain reaction in blood samples (serum or leukocytes) was used to monitor active CMV, HHV-6 and HHV-7 infections and disease in forty-three HSCT patients for up to 150 days after transplant. All adult recipients with a risk for CMV disease (D+/R+; D+/R-) were enrolled in this study. Acyclovir was used at low doses prior to the transplant as herpes virus prophylactic therapy. Patients who were at least 2 consecutive N-PCR positive for CMV received preemptive therapy with ganciclovir. The prevalence’s of positive active CMV, HHV-6 and HHV-7 infections were 72%, 4.6% and 13.9%, respectively. Thirteen patients died (30.2%). Biopsies confirmed CMV disease occurred in 8 out of 43 patients (18.6%), in the gastrointestinal tract. All of them presented active CMV infection and one presented active CMV+HHV-6 infection. None of these patients presented active HHV-7 infection. One patient with CMV disease died by disseminated CMV. Detection of active HHV-6 and HHV-7 infections was low and clinically significant complications were rare. CMV disease remains the most prominent disease associated with HSCT. Results show that surveillance with N-PCR ─ a sensitive, non-invasive and low-cost technique for detection of active beta herpesvirus infections ─ can be used when antigenemia or DNA quantitative methods are unavailable, because patients with a propensity for developing CMV disease can be readily identified and pre-emptive therapy started.