A new HPLC-UV method compared with HPLC-MS for daptomycin levels in human plasma samples

Application of a new HPLC-UV method on 122 plasma samples of patients from various AOUP wards, after analytical method settings, validation and comparison with a reference LC-MS/MS method. All research activities were performed according to the “Daptolin” protocol (approved by the Pisa University Hospital Ethics Committee, prot. num. 55945

Augmentation of Clozapine with Aripiprazole in Severe Psychotic Bipolar and Schizoaffective Disorders: A Pilot Study

AIM: To evaluate the efficacy and safety of the augmentation of clozapine with aripiprazole in patients with treatment-resistant schizoaffective and psychotic bipolar disorders in a retrospective manner. Pharmacodynamic and pharmacokinetic interactions between the two drugs were also investigated. PATIENTS: Three men and 4 women (median age 36 and 40 years, respectively) who had mean scores at BPRS and CGI-Severity of 59.1+/-12.0 and 5.4+/-0.5, respectively, were treated with clozapine (mean dose 292.9+/-220.7 mg/day). Patients received an adjunctive treatment with aripiprazole (mean dose 6.8 +/- 3.7 mg/day). Clozapine, norclozapine and aripiprazole plasma levels were measured by means of a high performance liquid chromatograpy with UV detection. RESULTS: Total scores at BPRS decreased significantly (from 59.1+/-12.0 to 51.1+/-15.6, p=0.007) after aripirazole augmentation. In particular, the factors "thought disorder" (from 10.4+/-4.4 to 9.0+/-4.5, p=.047) and "anergia" (from 10.0+/-2.7 to 8.0+/-2.4, p=.018) significantly improved. Concomitant administration of aripiprazole and clozapine did not result in an increase in side effects over the period of treatment. Dose-normalized plasma levels of both clozapine and norclozapine and the clozapine/norclozapine metabolic ratio in all patients did not vary as well. CONCLUSION: The augmentation of clozapine with aripirazole was safe and effective in severe psychotic schizoaffective and bipolar disorders which failed to respond to atypical antipsychotics. A possible pharmacokinetic interaction between clozapine and aripiprazole does not account for the improved clinical benefit obtained after aripiprazole augmentation

Effective, Robust Design of Community Mitigation for Pandemic Influenza: A Systematic Examination of Proposed US Guidance

BACKGROUND: The US government proposes pandemic influenza mitigation guidance that includes isolation and antiviral treatment of ill persons, voluntary household member quarantine and antiviral prophylaxis, social distancing of individuals, school closure, reduction of contacts at work, and prioritized vaccination. Is this the best strategy combination? Is choice of this strategy robust to pandemic uncertainties? What are critical enablers of community resilience? METHODS AND FINDINGS: We systematically simulate a broad range of pandemic scenarios and mitigation strategies using a networked, agent-based model of a community of explicit, multiply-overlapping social contact networks. We evaluate illness and societal burden for alterations in social networks, illness parameters, or intervention implementation. For a 1918-like pandemic, the best strategy minimizes illness to <1% of the population and combines network-based (e.g. school closure, social distancing of all with adults' contacts at work reduced), and case-based measures (e.g. antiviral treatment of the ill and prophylaxis of household members). We find choice of this best strategy robust to removal of enhanced transmission by the young, additional complexity in contact networks, and altered influenza natural history including extended viral shedding. Administration of age-group or randomly targeted 50% effective pre-pandemic vaccine with 7% population coverage (current US H5N1 vaccine stockpile) had minimal effect on outcomes. In order, mitigation success depends on rapid strategy implementation, high compliance, regional mitigation, and rigorous rescinding criteria; these are the critical enablers for community resilience. CONCLUSIONS: Systematic evaluation of feasible, recommended pandemic influenza interventions generally confirms the US community mitigation guidance yields best strategy choices for pandemic planning that are robust to a wide range of uncertainty. The best strategy combines network- and case-based interventions; network-based interventions are paramount. Because strategies must be applied rapidly, regionally, and stringently for greatest benefit, preparation and public education is required for long-lasting, high community compliance during a pandemic

Containing the accidental laboratory escape of potential pandemic influenza viruses

BACKGROUND: The recent work on the modified H5N1 has stirred an intense debate on the risk associated with the accidental release from biosafety laboratory of potential pandemic pathogens. Here, we assess the risk that the accidental escape of a novel transmissible influenza strain would not be contained in the local community. METHODS: We develop here a detailed agent-based model that specifically considers laboratory workers and their contacts in microsimulations of the epidemic onset. We consider the following non-pharmaceutical interventions: isolation of the laboratory, laboratory workers’ household quarantine, contact tracing of cases and subsequent household quarantine of identified secondary cases, and school and workplace closure both preventive and reactive. RESULTS: Model simulations suggest that there is a non-negligible probability (5% to 15%), strongly dependent on reproduction number and probability of developing clinical symptoms, that the escape event is not detected at all. We find that the containment depends on the timely implementation of non-pharmaceutical interventions and contact tracing and it may be effective (>90% probability per event) only for pathogens with moderate transmissibility (reproductive number no larger than R(0) = 1.5). Containment depends on population density and structure as well, with a probability of giving rise to a global event that is three to five times lower in rural areas. CONCLUSIONS: Results suggest that controllability of escape events is not guaranteed and, given the rapid increase of biosafety laboratories worldwide, this poses a serious threat to human health. Our findings may be relevant to policy makers when designing adequate preparedness plans and may have important implications for determining the location of new biosafety laboratories worldwide

A case report of a TDM-guided optimization of mitotane for a safe and effective long-term treatment

A 43-years old woman was diagnosed an adrenocortical carcinoma (AC) that was excised, whereas two lung metastases were un-operable. Mitotane 6 g/day was started as standard therapy but it was responsible for severe central nervous system (CNS) and gastrointestinal toxicities associated with a 10 kg body weight loss. A therapeutic drug monitoring (TDM) protocol demonstrated that mitotane plasma concentrations (>30 mg/L) exceeded the therapeutic range (14–20 mg/L) and increased even when drug daily dose was reduced by 50%. The increase in drug plasma concentrations was probably due to body slimming. Under continuous TDM control, a reduced mitotane dose (1.5 g/day) was definitively administered and it proved to be tolerable and effective. Indeed, lung metastases were excised and two years later there was no evidence of other neoplastic lesions. In conclusion, the adoption of therapeutic mitotane monitoring allowed the treatment of an AC patient with a reduced, tolerable and effective dose

Determination of Mitotane and principal metabolite by a simple HPLC-UV method and its validation in human plasma sample

Introduction Mitotane (DDD) is prescribed to inoperable adrenocortical renal carcinoma and Cushing’s syndrome. DDD and its principal metabolite, dichlorodiphenylethene (DDE), can accumulate in fat tissues and their plasma concentrations are associated with clinical improvement more than those of dichlorodiphenylacetate (DDA). The therapeutic range of plasma concentrations is 14–20 mg/L. Therefore, therapeutic drug monitoring (TDM) is required to exploit the maximum therapeutic benefit with acceptable toxicity. Methods Chromatographic conditions: stationary phase was represented by a Higgins Analytical C18 5 μm column (250mmx4.6 mm), maintained at 35 oC. Mobile phase was made by H2O/acetonitrile (10/90, v/v), and pumped at 1.0 ml/min. Peaks of interest were monitored at wavelength of 226 nm. Human plasma samples (200 μl) were added with aldrin (as Internal Standard) and 200 μl of acetonitrile for protein precipitation. Clear supernatants (50 μl) were injected within the HPLC apparatus. Results The average recovery of analytes was 95%, and the method was linear (r2=0.9988 and 0.9964 for DDD and DDE, respectively) within the range 1-40 mg/L. The values of limit of quantitation and detection were 0.2 mg/L and 0.3 mg/L for DDD and 0.066 mg/L and 0.099 mg/L for DDE, respectively. It is worth noting that sample preparation and run time are short enough to allow the analysis of at least 3 samples per hour (20 min total run). Indeed, the retention time (RT) of DDD and DDE are 7.06 min and 9.42 min, respectively, while the RT of the internal standard is 12.67 min. Finally, the method validation completely fulfilled FDA guidelines. Conclusions A reliable and rapid HPLC-UV method was developed for the measurement of mitotane and DDE concentrations in plasma samples using aldrin as internal standard for better accuracy and precision over the range of drug concentrations expected after the administration of mitotane at standard doses

A new validated HPLC-UV method for therapeutic monitoring of daptomycin in comparison with reference LC-MS/MS

Daptomycin, a cyclic lipopeptide antibiotic with broad spectrum of activity against Grampositive bacteria is also active against multi-resistant bacterial strains, as well as methicillinresistant S. aureus A or penicillin-resistant S. pneumoniae [1,2]. For these reasons, it is a viable alternative for treatment of persisting infections. However, the therapeutic drug monitoring of daptomycin is recommended because the known variability in drug disposition and the severe clinical conditions of patients [3]. Therefore, we developed a simple and fast UV-HPLC method according to FDA guidelines to monitor plasma concentrations of the drug and compared with LC-MS/MS gold standard method. After a liquid-liquid extraction, plasma calibration samples, quality controls and patients’ samples were injected in a HPLC instrument daptomycin and gentamicin (internal standard) that were resolved by a C18 250 × 4.6 mm, 5 µm stationary phase and peaks were monitored at UV = 262 nm. Mobile phase (isocratic flow of 1 mL/min) consisted of acetonitrile-buffer (KH2 PO4 20 mM pH = 3.2) 46:54, vol/vol. Under these conditions, IS and daptomycin peaked at 4.1 and 5.8 min after injection. Values of limits of detection and quantitation accounted for 1.65 and 5.00 (µg/ml), respectively. Values of method linearity (r2) in range 5−100 mg/L were 0.9975 and 0.9956 plasma samples and solvent standard, respectively. Inter- and intra-day variability coefficients were lower than 15 %. Method was applied to 122 patient plasma samples (r2=0.9474) and the output obtained (chromatographic peaks) has been processed with an algorithm (patent protected®) which allows to resolve any interfering peaks with analytes and obtain concentrations not significantly different from LC–MS/MS. These interfering peaks were processed also with other methods (“split peak” and “valley-valley”) commercially available but results were significantly different from those obtained with LC-MS/MS. In conclusion, the present method is demonstrated to be reliable and suitable for daptomycin TDM in clinical routine

Determination of mitotane and metabolite by a simple HPLC-UV method and application in plasma samples

Mitotane (dichlorodifenyldichloroethane, DDD) it is a polychlorinated compound derivative of dichlorodiphenyltrichloroethane (DDT), prescribed in inoperable adrenocortical renal carcinoma and Cushing’s syndrome [1,2]. DDD and its principal metabolite, dichlorodiphenylethene (DDE), can accumulate in fat tissues [3] and plasma concentrations are more related with clinical improvement, than those of dichlorodiphenylacetate (DDA), another metabolite of DDD. HPLC methods constitute a valid alternative to gas chromatography, and plasma concentrations of 14–20 mg/L are considered therapeutic concentrations. The stationary phase was represented by a Higgins Analytical C18 5 μm column (250mmx4.6 mm), at 35 oC. Mobile phase was made by H2O/acetonitrile (10/90, v/v) at 1.0 ml/min. Peaks of interest were monitored at wavelength of 226 nm. Human plasma sample preparation: 200 μl of plasma sample was added with aldrin (as Internal Standard) and 200 μl of acetonitrile for protein precipitation. Samples were vortexed for 30 sec and centrifuged at 12000 rpm x 10 minutes. Clear supernatants (50 μl) were injected in HPLC system. Average recovery was 95%, and the method was linear (r2=0.9988 and 0.9964 for DDD and DDE, respectively) within the range 1-40 mg/L. The LOQ and were 0.2 mg/L and 0.3 mg/L for DDD and 0.066 mg/L and 0.099 mg/L for DDE, respectively. Indeed, the retention time (RT) of DDD and DDE are 7.06 min and 9.42 min and 12.67 min for Internal Standard. Method was validated and applied to 30 plasma samples of 5 different patients affected by adrenocortical renal carcinoma. We analyzed the fluctuations of concentrations over time. In 30 plasma dosages, only 4 samples were within the range of 14-20 mg/L. The mitotane plasma level correlate with plasma DDE levels, according to pearson index. In conclusion, a reliable and rapid HPLC-UV method was developed for the measurement of mitotane and DDE concentrations in plasma samples using aldrin as internal standar

Determination of mitotane and principal metabolite by a simple HPLC-UV method and its validation in human plasma sample

NTRODUCTION Mitotane (dichlorodifenyldichloroethane, DDD) it is a polychlorinated compound derivative of dichlorodiphenyltrichloroethane (DDT) that is prescribed to in inoperable adrenocortical renal carcinoma and Cushing’s syndrome (1,2). DDD and its principal metabolite, dichlorodiphenylethene (DDE), can accumulate in fat tissues (3) and their plasma concentrations are more related with clinical improvement, than those of dichlorodiphenylacetate (DDA), another metabolite of DDD (4). Therapeutic monitoring of plasma concentrations is thus required to combine good therapeutic efficacy with acceptable toxicity. HPLC methods constitute a valid alternative to gas chromatography, and plasma concentrations of 14–20 mg/L are considered therapeutic concentrations. MATERIALS AND METHODS Chromatographic conditions: stationary phase was represented by a Higgins Analytical C18 5 μm column (250mmx4.6 mm), maintained at 35 oC. Mobile phase was made by H2O/acetonitrile (10/90, v/v) and pumped at flow of 1.0 ml/min. Peaks of interest were monitored at wavelenght of 226 nm. Human plasma sample preparation: 200 μl of plasma sample was added with aldrin (as Internal Standard) and 200 μl of acetonitrile for protein precipitation. Samples were vortexed for 30 sec and centrifuged at 12000 rpm x 10 minutes. Clear supernatants (50 μl) were injected within the HPLC apparatus. RESULTS The average recovery of analytes was 95%, and the method was linear (r2=0.9988 and 0.9964 for DDD and DDE, respectively) within the range 1-40 mg/L. The values of limit of quantitation and detection were 0.2 mg/L and 0.3 mg/L for DDD and 0.066 mg/L and 0.099 mg/L for DDE, respectively. It is worth noting that sample preparation and run time are short enough to allow the analysis of at least 4 samples per hour (15 min total run). Indeed, the retention time (RT) of DDD and DDE are 7.06 min and 9.42 min, respectively, while the RT of the internal standard is 12.67 min. Finally, the validation process returned inter- and intra-day mean accuracies (1.30% and 1.60%, respectively) and precision (6.45% and 7.70%, respectively) within the limit of FDA guidelines. Therefore, the method has been adopted at the TDM Lab of Clinical Pharmacology Unit, University Hospital, Pisa, for routine monitoring of mitotane plasma concentrations in patients affectd by adrenocortical renal carcinoma. References: [1] B. Allolio, et al. J of Clin Endocrinol &amp; Metab., 91(6), 2027-2037 (2006). [2] P. Kamenicky, et al. J of Clin Endocrinol &amp; Metab., 96(9), 2796-2804 (2011). [3] S. Kitamura, et al. Drug metabolism and disposition, 30, 113-118 (2002). [4] A. kasperlik-Zaluska, et al. J of Exp Ther &amp; Oncology, 5, 125-132 (2005)