Maria Pieri

Analytical and Pharmacological Aspects of Therapeutic Drug Monitoring of mTOR Inhibitors

Mammalian Target Of Rapamycin (mTOR) inhibitors represent a new class of immunosuppressant drugs extensively used for the prevention and the treatment of graft rejection in organ transplant recipients. Their current use is due to referred low nephrotoxic effects, particularly important in kidney transplanted and/or patients with renal failure. The most representative drugs of such class are Sirolimus (Siro) and Everolimus (Rad). Both drugs show a narrow therapeutic window, therefore, monitoring of whole-blood drug levels is recommended in order to optimize the therapy. Among the available assays, Liquid Chromatography coupled with UltraViolet or Electrospray Tandem Mass Spectrometry methods (LC/UV or LC/ESI-MSMS) are the most accurate and specific ones. A reliable alternative is represented by immunoassays, which offer the opportunity to minimize sample pre-treatment, thus reducing the time between drawing blood sample and measuring the drug concentration, an important aspect in high-throughput analyses. Despite this, a limitation in the use of immunoassays for therapeutic drug monitoring is the lower specifity compared with the chromatographic methods when analysing structurally-related drugs. New insights to optimize mTOR inhibitors regimens seem to be offered by the evaluation of CYP450 3A activity by using the probe drug approach. To such purpose, there are a number of major probe drugs used for in vivo studies including: midazolam, cortisol, lidocaine, nifedipine, dextromethorphan, erythromycin, dapsone and alfentanil. The aim of the present paper is to report the most recent knowledge concerning this issue, supplying a critical and comprehensive review for whom are involved both in the clinical and analytical areas.

Determination of urinary S-phenylmercapturic acid by liquid chromatography-tandem mass spectrometry

Urinary S-phenylmercapturic acid (S-PMA) is considered a useful biomarker for the measurement of low levels of benzene exposure, related to occupational exposure, smoking habits or environmental pollution. S-PMA quantitative analysis requires highly sensitive and specific techniques and purification procedures, mainly based on liquid-liquid or solid-phase extraction, which result in time expensive analyses. A method was developed for the quantitative determination of S-PMA in urine by using a simple, reproducible and easily automatizable HPLC purification followed by LC/ESI-NI/MS2 analysis. In order to reduce the cost of the analysis, related to the use of expensive labeled standards, p-bromo-S-phenylmercapturic acid (p-Br-S-PMA) was synthesized, characterized and used as internal standard. The feasibility and efficacy of the proposed method were examined by constructing calibration curves in the range from 6.2 to 200 microg/l and data were analyzed in terms of linearity and statistical parameters. The detection limit, related to the purification of 1 ml urine sample is 5 microg/l. The method was applied to the analysis of 12 urine samples from smoker subjects non-occupationally exposed to benzene. S-PMA urinary levels ranged from 13.6 to >200 microg/l, suggesting a high influence of life style in the S-PMA excretion. The proposed analytical method is suitable for the biological monitoring of both smoker and non-smoker workers, occupationally exposed to benzene. By processing at least 2 ml of urine samples, the method appears to be also useful for the evaluation of benzene uptake due to the environmental pollution.

Screening of several drugs of abuse in Italian workplace drug testing: performance comparisons of on-site screening tests and a fluorescence polarization immunoassay-based device.

According to the Italian laws, some categories of workers entrusted with duties possibly constituting a threat to security, physical safety, and health of third parties have to be screened to exclude the use/abuse of the following drugs of abuse: opiates, cocaine, cannabinoids, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methadone, and buprenorphine. Toxicological tests can be performed with urinary on-site rapid screening devices, provided that sensitivities up to specified cutoffs are ensured. The present study reports performances, in terms of sensitivity, specificity, and accuracy, of an automatic on-site test and of an FPIA-based device, using gas chromatography/mass spectrometry (GC/MS) as a reference methodology. Three levels of concentration were tested, corresponding to the cutoff and to 2 and 3 times the limits, respectively. In terms of sensitivities, neither the on-site nor the benchtop instrumentations gave positive results, since values of zero percentage were obtained for concentrations up to 2-fold the limits. Even if good results were obtained in terms of specificity and accuracy by both devices, none of them seem to be adequate for the current application to the toxicological screening at workplaces. In fact, a rapid screening device can be used for drug tests provided that it ensures sensitivity at the prescribed cutoffs. Data showed that such is completely rejected and a more sensitive instrumentation should be preferred.

Immunoassay determination of rapamycin: reliability of the method with respect to liquid chromatography mass spectrometric quantification.

Abstract:  Immunochemical assays represent a promising tool for quantification of immunosuppressants in organ transplanted patients, because they require small sample volumes and minimum sample pre‐treatment; nevertheless considerations about method specificity, sensitivity and reproducibility cannot be overlooked. The present paper investigates the reliability of using the immunoparticle enzyme immunoassay (MEIA) for the quantification of blood rapamycin (RAPA) levels in therapeutic drug monitoring of renal transplanted patients with respect to a validated liquid chromatography tandem mass spectrometric (LC/ESI‐MSMS) method, used as reference. Linearity of MEIA was tested over the range 0.0–30.0 ng/mL, with accuracy and precision within acceptable limits. Fifty‐two blood samples were collected from 42 renal transplanted patients and analyzed simultaneously by both methods. The Pearson’s regression analysis gave the following parameters: correlation equation [RAPA]MEIA = 1.330 + 0.776 [RAPA]LC/ESI‐MSMS, r = 0.8526, SD = 1.778, p < 0.0001. The obtained average rapamycin concentration was 8.8 ± 3.4 ng/mL using MEIA and 9.6 ± 3.7 ng/mL for LC/ESI‐MSMS, with an overall underestimation of about 6% of the immunoenzymatic test. Accuracy of MEIA ranged from −33% to 36% with respect to the reference mass spectrometric method. Although immunoenzymatic test represents a fast and sufficiently accurate method for its use in clinical practice, specificity of the assay is still not sufficiently investigated and reference methods and/or Proficiency Testing Scheme should be used as external control.

Epirubicin permeation of personal protective equipment can induce apoptosis in keratinocytes

The present study investigated the epirubicin (EPI) permeability of various commercially available glove types, as well as toxicity mechanisms and effects on human keratinocyte cell line (HaCaT). Permeability experiments were carried out on various commercially available gloves, differing as regards material and thickness. Permeability was evaluated after different “contact times” and the influence of EPI solutions pH (acid and neutral) on permeability was also examined. Toxicity of EPI toward skin was tested by evaluating the effects of the drug on cell growth and apoptosis, by using an in vitro model based on cultured immortalized human keratinocytes. No permeation was detected in the case of EPI neutral solutions; in contrast, acid solutions were found to penetrate low thickness nitrile gloves. Obtained results also showed the induction of apoptosis in epithelial cells through the activation of intrinsic pathway p53-independent occurring even when cells are exposed at low drug concentration. EPI solutions pH influences the gloves permeability; once penetrated, EPI at concentrations lower than those able to penetrate the nitrile glove during the 8-h work-shift can cause apoptosis in epithelial cells. The findings reported here highly support the choice of either natural rubbers gloves or high thickness nitrile ones for preventing the occupational exposure to EPI.