Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods for the therapeutic drug monitoring of cytotoxic anticancer drugs: An update.

In the era of precision medicine, there is increasing evidence that conventional cytotoxic agents may be suitable candidates for therapeutic drug monitoring (TDM)- guided drug dosage adjustments and patient's tailored personalization of non-selective chemotherapies. To that end, many liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assays have been developed for the quantification of conventional cytotoxic anticancer chemotherapies, that have been comprehensively and critically reviewed. The use of stable isotopically labelled internal standards (IS) of cytotoxic drugs was strikingly uncommon, accounting for only 48 % of the methods found, although their use could possible to suitably circumvent patients' samples matrix effects variability. Furthermore, this approach would increase the reliability of cytotoxic drug quantification in highly multi-mediated cancer patients with complex fluctuating pathophysiological and clinical conditions. LC-MS/MS assays can accommodate multiplexed analyses of cytotoxic drugs with optimal selectivity and specificity as well as short analytical times and, when using stable-isotopically labelled IS for quantification, provide concentrations measurements with a high degree of certainty. However, there are still organisational, pharmacological, and medical constraints to tackle before TDM of cytotoxic drugs can be more largely adopted in the clinics for contributing to our ever-lasting quest to improve cancer treatment outcomes.

2019 update of the European AIDS Clinical Society Guidelines for treatment of people living with HIV version 10.0.

BACKGROUND: The European AIDS Clinical Society (EACS) Guidelines cover key aspects of HIV management with major updates every two years. GUIDELINE HIGHLIGHTS: The 2019 Guidelines were extended with a new section focusing on drug-drug interactions and other prescribing issues in people living with HIV (PLWH). The recommendations for treatment-naïve PLWH were updated with four preferred regimens favouring unboosted integrase inhibitors. A two-drug regimen with dolutegravir and lamivudine, and a three-drug regimen including doravirine were also added to the recommended initial regimens. Lower thresholds for hypertension were expanded to all PLWH and for cardiovascular disease prevention, the 10-year predicted risk threshold for consideration of antiretroviral therapy (ART) modification was lowered from 20% to 10%. Frailty and obesity were added as new topics. It was specified to use urine albumin to creatinine ratio to screen for glomerular disease and urine protein to creatinine ratio for tubular diseases, and thresholds were streamlined with the Kidney Disease: Improving Global Outcomes (KDIGO) recommendations. Hepatitis C virus (HCV) treatment recommendations were split into preferred and alternative treatment options. The algorithm for management of recently acquired HCV infection was updated and includes recommendations for early chronic infection management. Treatment of resistant tuberculosis (TB) was streamlined with the World Health Organization (WHO) recommendations, and new tables on immune reconstitution inflammatory syndrome, on when to start ART in the presence of opportunistic infections and on TB drug dosing were included. CONCLUSIONS: The EACS Guidelines underwent major revisions of all sections in 2019. They are available in four different formats including a new interactive web-based version and are translated into Chinese, French, German, Japanese, Portuguese, Russian and Spanish.

Potentially inappropriate medications in older adults living with HIV.

OBJECTIVES: We assessed the prevalence of potentially inappropriate medication (PIM) among older (≥ 65 years) people living with HIV (O-PLWH) in the region of Madrid. METHODS: We analysed the dispensation registry of community and hospital pharmacies from the Madrid Regional Health Service (SERMAS) for the period between 1 January and 30 June 2017, looking specifically at PIMs according to the 2019 Beers criteria. Co-medications were classified according to the Anatomical Therapeutic Chemical (ATC) classification system. RESULTS: A total of 6 636 451 individuals received medications. Of these individuals, 22 945 received antiretrovirals (ARVs), and of these 1292 were O-PLWH. Overall, 1135 (87.8%) O-PLWH were taking at least one co-medication, and polypharmacy (at least five co-medications) was observed in 852 individuals (65.9%). A PIM was identified in 482 (37.3%) O-PLWH. Factors independently associated with PIM were polypharmacy [adjusted odds ratio (aOR) 7.08; 95% confidence interval (CI) 5.16-9.72] and female sex (aOR 1.75; 95% CI 1.30-2.35). The distribution of PIMs according to ATC drug class were nervous system drugs (n = 369; 28.6%), musculoskeletal system drugs (n = 140; 10.8%), gastrointestinal and metabolism drugs (n = 72; 5.6%), cardiovascular drugs (n = 61; 4.7%), respiratory system drugs (n = 13; 1.0%), antineoplastic and immunomodulating drugs (n = 10; 0.8%), and systemic anti-infectives (n = 2; 0.2%). Five drugs accounted for 84.8% of the 482O PLWH with PIMs: lorazepam (38.2%), ibuprofen (18.0%), diazepam (10.2%), metoclopramide (9.9%), and zolpidem (8.5%). CONCLUSIONS: Prescription of PIMs is highly prevalent in O-PLWH. Consistent with data in uninfected elderly people, the most frequently observed PIMs were benzodiazepines and nonsteroidal anti-inflammatory drugs . Targeted interventions are warranted to reduce inappropriate prescribing and polypharmacy in this vulnerable population.

Prevalence and management of drug-drug interactions with antiretroviral treatment in 2069 people living with HIV in rural Tanzania: a prospective cohort study.

OBJECTIVES: Widespread access to antiretroviral therapy (ART) has substantially increased life expectancy in sub-Saharan African countries. As a result, the rates of comorbidities and use of co-medications among people living with HIV are increasing, necessitating a sound understanding of drug-drug interactions (DDIs). We aimed to assess the prevalence and management of DDIs with ART in a rural Tanzanian setting. METHODS: We included consenting HIV-positive adults initiating ART in the Kilombero and Ulanga Antiretroviral Cohort (KIULARCO) between January 2013 and December 2016. DDIs were classified using www.hiv-druginteractions.org as red (contra-indicated), amber (potential clinical relevance requiring dosage adjustment/monitoring), yellow (weak clinical significance unlikely to require further management) or green (no interaction). We assessed management of amber DDIs by evaluating monitoring of laboratory or clinical parameters, or changes in drug dosages. RESULTS: Of 2069 participants, 1945 (94%) were prescribed at least one co-medication during a median follow-up of 1.8 years. Of these, 645 (33%) had at least one potentially clinically relevant DDI, with the highest grade being red in nine (< 1%) and amber in 636 (33%) participants. Of the 23 283 prescriptions, 19 (< 1%) and 1745 (7%) were classified as red and amber DDIs, respectively. Overall, 351 (2%) prescriptions were red DDIs or not appropriately managed amber DDIs. CONCLUSIONS: Co-medication use was common in this rural sub-Saharan cohort. A third of participants had DDIs requiring further management. Of the 9% of participants with not appropriately managed DDIs, most were with cardiovascular and analgesic drugs. This highlights the importance of physicians' awareness of DDIs for their recognition and management.

Pharmacokinetics, Placental and Breast Milk Transfer of Antiretroviral Drugs in Pregnant and Lactating Women Living with HIV.

BACKGROUND: Remarkable progress has been achieved in the identification of HIV infection in pregnant women and in the prevention of vertical HIV transmission through maternal antiretroviral treatment (ART) and neonatal antiretroviral drug (ARV) prophylaxis in the last two decades. Millions of women globally are receiving combination ART throughout pregnancy and breastfeeding, periods associated with significant biological and physiological changes affecting the pharmacokinetics (PK) and pharmacodynamics (PD) of ARVs. The objective of this review was to summarize currently available knowledge on the PK of ARVs during pregnancy and transport of maternal ARVs through the placenta and into the breast milk. We also summarized main safety considerations for in utero and breast milk ARVs exposures in infants. METHODS: We conducted a review of the pharmacological profiles of ARVs in pregnancy and during breastfeeding obtained from published clinical studies. Selected maternal PK studies used a relatively rich sampling approach at each ante- and postnatal sampling time point. For placental and breast milk transport of ARVs, we selected the studies that provided ratios of maternal to the cord (M:C) plasma and breast milk to maternal plasma (M:P) concentrations, respectively. RESULTS: We provide an overview of the physiological changes during pregnancy and their effect on the PK parameters of ARVs by drug class in pregnancy, which were gathered from 45 published studies. The PK changes during pregnancy affect the dosing of several protease inhibitors during pregnancy and limit the use of several ARVs, including three single tablet regimens with integrase inhibitors or protease inhibitors co-formulated with cobicistat due to suboptimal exposures. We further analysed the currently available data on the mechanism of the transport of ARVs from maternal plasma across the placenta and into the breast milk and summarized the effect of pregnancy on placental and of breastfeeding on mammal gland drug transporters, as well as physicochemical properties, C:M and M:P ratios of individual ARVs by drug class. Finally, we discussed the major safety issues of fetal and infant exposure to maternal ARVs. CONCLUSIONS: Available pharmacological data provide evidence that physiological changes during pregnancy affect maternal, and consequently, fetal ARV exposure. Limited available data suggest that the expression of drug transporters may vary throughout pregnancy and breastfeeding thereby possibly impacting the amount of ARV crossing the placenta and secreted into the breast milk. The drug transporter's role in the fetal/child exposure to maternal ARVs needs to be better understood. Our analysis underscores the need for more pharmacological studies with innovative study design, sparse PK sampling, improved study data reporting and PK modelling in pregnant and breastfeeding women living with HIV to optimize their treatment choices and maternal and child health outcomes.

Revisiting systemic treatment of bacterial endophthalmitis: a review of intravitreal penetration of systemic antibiotics.

BACKGROUND: Adjunctive systemic antibiotic therapy for treatment of bacterial endophthalmitis is controversial but common practice due to the severity of the disease. In the absence of guidance documents, several antibiotic regimens are being used without applying evidence-based prescribing, leading to inappropriate treatment of this serious eye condition. OBJECTIVES: To summarize available data on intravitreal penetration of systemically administered antibiotics and to discuss their usefulness from a microbiological and pharmacological point of view. SOURCES: We performed a systematic PubMed search of studies investigating antibiotic concentrations in the vitreous after systemic administration in humans, and selected animal models. CONTENT: The best-documented agents achieving therapeutic levels in the vitreous are meropenem, linezolid and moxifloxacin. Vancomycin, cefazoline, ceftriaxone, ceftazidime, imipenem and trimethoprim-sulfamethoxazole reach levels justifying their use in specific situations. Available data do not support the use of ciprofloxacin, levofloxacin, aminoglycosides, aminopenicillins, piperacillin, cefepime and clarithromycin. With very limited but available promising data, the use of daptomycin and rifampicin deserves further investigation. IMPLICATIONS: The choice of the adjunctive systemic antibiotic agent-in situations where it is considered relevant for treatment-must to date be made on an individual basis, considering microbiological aspects as well as operative status and inflammation of the eye. This review gives a systematic overview of antibiotic options and provides guidance to the clinician striving for optimal systemic antibiotic treatment of bacterial endophthalmitis.

Highlights of the 2017 European AIDS Clinical Society (EACS) Guidelines for the treatment of adult HIV-positive persons version 9.0.

BACKGROUND: The European AIDS Clinical Society (EACS) Guidelines have since 2005 provided multidisciplinary recommendations for the care of HIV-positive persons in geographically diverse areas. GUIDELINE HIGHLIGHTS: Major revisions have been made in all sections of the 2017 Guidelines: antiretroviral treatment (ART), comorbidities, coinfections and opportunistic diseases. Newly added are also a summary of the main changes made, and direct video links to the EACS online course on HIV Management. Recommendations on the clinical situations in which tenofovir alafenamide may be considered over tenofovir disoproxil fumarate are provided, and recommendations on which antiretrovirals can be used safely during pregnancy have been revised. Renal and bone toxicity and hepatitis C virus (HCV) treatment have been added as potential reasons for ART switches in fully virologically suppressed individuals, and dolutegravir/rilpivirine has been included as a treatment option. In contrast, dolutegravir monotherapy is not recommended. New recommendations on non-alcoholic fatty liver disease, chronic lung disease, solid organ transplantation, and prescribing in elderly are included, and human papilloma virus (HPV) vaccination recommendations have been expanded. All drug-drug interaction tables have been updated and new tables are included. Treatment options for direct-acting antivirals (DAAs) have been updated and include the latest combinations of sofosbuvir/velpatasvir/voxilaprevir and glecaprevir/pibrentasvir. Recommendations on management of DAA failure and acute HCV infection have been expanded. For treatment of tuberculosis (TB), it is underlined that intermittent treatment is contraindicated, and for resistant TB new data suggest that using a three-drug combination may be as effective as a five-drug regimen, and may reduce treatment duration from 18-24 to 6-10 months. CONCLUSIONS: Version 9.0 of the EACS Guidelines provides a holistic approach to HIV care and is translated into the six most commonly spoken languages.

Health care provider communication training in rural Tanzania empowers HIV-infected patients on antiretroviral therapy to discuss adherence problems.

OBJECTIVES: Self-reported adherence assessment in HIV-infected patients on antiretroviral therapy (ART) is challenging and may overestimate adherence. The aim of this study was to improve the ability of health care providers to elicit patients' reports of nonadherence using a "patient-centred" approach in a rural sub-Saharan African setting. METHODS: A prospective interventional cohort study of HIV-infected patients on ART for ≥ 6 months attending an HIV clinic in rural Tanzania was carried out. The intervention consisted of a 2-day workshop for health care providers on patient-centred communication and the provision of an adherence assessment checklist for use in the consultations. Patients' self-reports of nonadherence (≥ 1 missed ART dose/4 weeks), subtherapeutic plasma ART concentrations (< 2.5th percentile of published population-based pharmacokinetic models), and virological and immunological failure according to the World Health Organization definition were assessed before and after (1-3 and 6-9 months after) the intervention. RESULTS: Before the intervention, only 3.3% of 299 patients included in the study reported nonadherence. Subtherapeutic plasma ART drug concentrations and virological and immunological failure were recorded in 6.5%, 7.7% and 14.5% of the patients, respectively. Two months after the intervention, health care providers detected significantly more patients reporting nonadherence compared with baseline (10.7 vs. 3.3%, respectively; P < 0.001), decreasing to 5.7% after 6-9 months. A time trend towards higher drug concentrations was observed for efavirenz but not for other drugs. The virological failure rate remained unchanged whereas the immunological failure rate decreased from 14.4 to 8.7% at the last visit (P = 0.002). CONCLUSIONS: Patient-centred communication can successfully be implemented with a simple intervention in rural Africa. It increases the likelihood of HIV-infected patients reporting problems with adherence to ART; however, sustainability remains a challenge.

Emergence of Acquired HIV-1 Drug Resistance Almost Stopped in Switzerland: A 15-Year Prospective Cohort Analysis.

BACKGROUND: Drug resistance is a major barrier to successful antiretroviral treatment (ART). Therefore, it is important to monitor time trends at a population level. METHODS: We included 11 084 ART-experienced patients from the Swiss HIV Cohort Study (SHCS) between 1999 and 2013. The SHCS is highly representative and includes 72% of patients receiving ART in Switzerland. Drug resistance was defined as the presence of ≥1 major mutation in a genotypic resistance test. To estimate the prevalence of drug resistance, data for patients with no resistance test was imputed based on the patient's risk of harboring drug-resistant viruses. RESULTS: The emergence of new drug resistance mutations declined dramatically from 401 to 23 patients between 1999 and 2013. The upper estimated prevalence limit of drug resistance among ART-experienced patients decreased from 57.0% in 1999 to 37.1% in 2013. The prevalence of 3-class resistance decreased from 9.0% to 4.4% and was always <0.4% for patients who initiated ART after 2006. Most patients actively participating in the SHCS in 2013 with drug-resistant viruses initiated ART before 1999 (59.8%). Nevertheless, in 2013, 94.5% of patients who initiated ART before 1999 had good remaining treatment options based on Stanford algorithm. CONCLUSIONS: Human immunodeficiency virus type 1 drug resistance among ART-experienced patients in Switzerland is a well-controlled relic from the era before combination ART. Emergence of drug resistance can be virtually stopped with new potent therapies and close monitoring.

Pharmacokinetic and pharmacodynamic analysis of efavirenz dose reduction using an in vitro-in vivo extrapolation model.

The pharmacokinetics (PK) of efavirenz (EFV) is characterized by marked interpatient variability that correlates with its pharmacodynamics (PD). In vitro-in vivo extrapolation (IVIVE) is a "bottom-up" approach that combines drug data with system information to predict PK and PD. The aim of this study was to simulate EFV PK and PD after dose reductions. At the standard dose, the simulated probability was 80% for viral suppression and 28% for central nervous system (CNS) toxicity. After a dose reduction to 400 mg, the probabilities of viral suppression were reduced to 69, 75, and 82%, and those of CNS toxicity were 21, 24, and 29% for the 516 GG, 516 GT, and 516 TT genotypes, respectively. With reduction of the dose to 200 mg, the probabilities of viral suppression decreased to 54, 62, and 72% and those of CNS toxicity decreased to 13, 18, and 20% for the 516 GG, 516 GT, and 516 TT genotypes, respectively. These findings indicate how dose reductions might be applied in patients with favorable genetic characteristics.

[Drug-drug interactions with HIV treatments]. / Medikamenteninteraktion bei HIV-Therapien.

We develop a differential diagnosis and diagnostic approach to a possible drug-drug interaction between a cART based on boosted atazanavir and newly given drugs. History taking should not only encompass prescribed but also over-the-counter medication. Exchange of information between attending physicians as to therapeutic changes, documented side effects and adherence, therapeutic drug monitoring of cART and evaluation by pharmacologist in complex situations are the diagnostic tools at hand.

Determination of the novel non-peptidic HIV-protease inhibitor tipranavir by HPLC-UV after solid-phase extraction.

An HPLC method previously described for the assay of amprenavir (APV), ritonavir (RTV), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV), nevirapine (NVP) and efavirenz (EFV) can be also conveniently applied, with minor gradient program adjustment, for the determination of the novel non-peptidic HIV protease inhibitor tipranavir (TPV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection (DAD). After viral inactivation by heat, the plasma is diluted with phosphate buffer (pH 7), and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with a solution of 0.1% H3PO4 solution neutralised to pH 7, and TPV is eluted with MeOH. The resulting eluate is evaporated and reconstituted in 100 microl MeOH/H2O 50/50. A 40 microl volume is injected onto a Nucleosil C18 AB column and TPV is analysed by UV detection at 201 nm using a gradient elution program constituted of MeCN and phosphate buffer adjusted to pH 5.12 and containing 0.02% sodium heptanesulfonate. The calibration curves are linear up to 75 microg/ml, with a lower limit of quantification of 0.125 microg/ml. The mean absolute recovery of TPV is 77.1+/-4.0%. The method is precise with mean inter-day coefficient of variations (CVs) within 2.2-3.4%, and accurate (range of inter-day deviations from 0.7 to 1.2%). The method has been validated and is currently applied to the monitoring of TPV plasma levels in HIV patients.

Determination of the new HIV-protease inhibitor atazanavir by liquid chromatography after solid-phase extraction.

An HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz is proposed here for the simultaneous analysis of the new HIV protease inhibitor atazanavir (ATV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection. After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl) with clozapine (internal standard) is diluted 1 + 1 with phosphate buffer pH 7 and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H(3)PO(4) neutralised with NaOH to pH 7. ATV is eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl MeOH/H(2)O 50/50. A 40 microl volume is injected onto a Nucleosil 100-5 microm C18 AB column. ATV is analysed by UV detection at 201 nm using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.14. The mobile phase also contains 0.02% sodium heptanesulfonate, enabling an excellent separation of ATV from the other HIV protease inhibitors (PIs) amprenavir, indinavir, saquinavir, ritonavir, lopinavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. The calibration curves are linear up to 10 microg/ml, with a lower limit of quantification of 0.2 microg/ml. The mean absolute recovery of ATV is 96.4 +/- 3.2%. The method is precise with mean inter-day CVs within 1.1-6.1%, and accurate (range of inter-day deviations +0.3 to +2.3%). The method has been validated and is currently applied to the monitoring of ATV in HIV patients.

Determination of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction: application for the assessment of their transplacental passage at delivery.

An adaptation of the HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz after solid-phase extraction is proposed here for the separate analysis of the newer PI lopinavir (LPV) and the NNRTI nevirapine (NVP). After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl), with clozapine added as internal standard, is diluted 1+1 with phosphate buffer pH 7 and subjected to a solid-phase extraction on a C(18) cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H(3)PO(4) neutralised with NaOH to pH 7. LPV and NVP are eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl MeOH 50%. A 40-microl volume is injected onto a Nucleosil 100, 5 microm C(18) AB column. LPV and NVP are analysed separately using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.07 and containing 0.02% sodium heptanesulfonate. LPV and NVP are detected by UV at 201 and 282 nm, respectively. The calibration curves are linear up to 10 microg/ml. The mean absolute recovery of LPV and NVP is 91% and 88%, respectively. The method is precise with mean inter-day C.V.s within 2.1-6.6% and 0.9-1.7% for LPV and NVP, and accurate (range of inter-day deviations -1.1 to +2.4%, and -1.9 to +0.8%, for LPV and NVP, respectively). The method has been validated and is currently applied to the monitoring of LPV and NVP in HIV patients, and has been notably applied in a study aimed at assessing the extent of transplacental passage of nevirapine and PIs, notably lopinavir, at the time of delivery in pregnant HIV-infected women.

Nelfinavir plasma levels under twice-daily and three-times-daily regimens: high interpatient and low intrapatient variability.

Nelfinavir has been recently approved as a twice-daily (BID) dose regimen, but no evaluation of the influence of this regimen change on patients' protease inhibitor exposure has been published. The aim of this study was to compare trough plasma concentrations of nelfinavir obtained under the 1250-mg b.i.d regimen with the levels achieved with the original 750-mg three-times-daily (TID) regimen in 56 HIV-infected patients. Blood samples were obtained at steady state before the morning dose of nelfinavir. Plasma levels were measured by high-performance liquid chromatography. Eleven and 45 patients were following TID and BID regimens, respectively. Trough concentrations ranged from 0.14 to 11.74 mg/L and from 0.36 to 10.57 mg/L under TID and BID regimens, respectively. Large interpatient (coefficient of variation: 153%) and modest intrapatient (45%) variabilities of nelfinavir levels were observed. Twenty-one patients (38%) and six patients (11%) had levels above and below, respectively, the trough nelfinavir range (1.0--3.0 mg/L) recommended by the manufacturer. Trough levels are not affected by the dosing regimen; they mainly reflect the important interindividual variability, while remaining fairly stable over time. Many subjects had plasma levels repeatedly outside the assumed therapeutic range. Dose adjustment based on therapeutic drug monitoring may contribute to optimizing antiretroviral therapy.

Efavirenz plasma levels can predict treatment failure and central nervous system side effects in HIV-1-infected patients.

OBJECTIVE: Limited information exists on the clinical usefulness of drug level monitoring for efavirenz, a once-daily non-nucleoside reverse transcriptase inhibitor (NNRTI). The aim of this study was to determine whether efavirenz plasma concentration monitoring could predict treatment failure and central nervous system (CNS) tolerability. METHODS: Blood samples were obtained from 130 HIV-infected patients receiving efavirenz in combination with other antiretroviral agents for more than 3 months. Efavirenz plasma concentrations were measured by high-performance liquid chromatography. An evaluation of CNS side-effects was performed and the viral load, CD4 cell count and other clinical and laboratory data were assessed. In 85 patients, these measures were repeated at 3 month intervals. RESULTS: Efavirenz plasma levels (n = 226) were measured at an average of 14 h after drug intake. Drug concentrations ranged from 125 to 15230 microg/l (median 2188). Large inter-patient (CV 118%) and limited intra-patient (CV 30%) variabilities were observed in efavirenz levels. Virological failure was observed in 50% of patients with low efavirenz levels (< 1000 microg/l) versus 22 and 18% in patients with 1000-4000 microg/l or more than 4000 microg/l, respectively. CNS toxicity was approximately three times more frequent in patients with high efavirenz levels (> 4000 microg/l) compared with patients with 1000-4000 microg/l. CONCLUSION: Treatment failure and CNS side-effects are associated with low and high efavirenz plasma levels, respectively. The important inter-individual variability in efavirenz levels strongly argues for dose adjustment on the basis of therapeutic drug monitoring to optimize treatment.

Simultaneous determination of the HIV protease inhibitors indinavir, amprenavir, saquinavir, ritonavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitor efavirenz by high-performance liquid chromatography after solid-phase extraction.

As part of an on-going study on the suitability of a formal therapeutic drug monitoring (TDM) of antiviral drugs for improving the management of HIV infection, a high-performance liquid chromatography method has been developed to quantify simultaneously in plasma five HIV protease inhibitors (PIs) (i.e., indinavir, amprenavir, saquinavir, ritonavir, nelfinavir) and the novel non-nucleoside reverse transcriptase inhibitor efavirenz. After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl), with clozapine added as internal standard, is diluted 1:1 with phosphate buffer, pH 7 and subjected to a solid-phase extraction on a C18 cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H3PO4 neutralised with NaOH to pH 7. PIs and efavirenz are eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl 50% MeOH. A 40-microl volume is subjected to HPLC analysis onto a Nucleosil 100, 5 microm C18 AB column, using a gradient elution of MeCN and phosphate buffer adjusted to pH 5.15 and containing 0.02% sodium heptanesulfonate: 15:85 at 0 min-->30:70 at 2 min-->32:68 at 8 min-->42:58 at 18 min-->46:54 at 34 min, followed by column cleaning with MeCN-buffer, pH 5.15 (90:10), onto which 0.3% AcOH is added. Clozapine, indinavir, amprenavir, saquinavir, ritonavir, efavirenz and nelfinavir are detected by UV at 201 nm at a retention time of 8.2, 13.0, 16.3, 21.5, 26.5, 28.7 and 31.9 min, respectively. The total run time for a single analysis is 47 min, including the washing-out and reequilibration steps. The calibration curves are linear over the range 100-10,000 ng/ml. The absolute recovery of PIs/efavirenz is always higher than 88%. The method is precise with mean inter-day relative standard deviations within 2.5-9.8% and accurate (range of inter-day deviations -4.6 to +4.3%). The in vitro stability of plasma spiked with PIs/efavirenz at 750, 3000 and 9000 ng/ml has been studied at room temperature, -20 degrees C and +60 degrees C. The method has been validated and is currently applied to the monitoring of PIs and efavirenz in HIV patients. This HPLC assay may help clinicians confronted to questionable compliance, side effects or treatment failure in elucidating whether patients are exposed to adequate circulating drug levels. The availability of such an assay represents an essential step in elucidating the utility of a formal TDM for the optimal follow-up of HIV patients.

Sequential administration of temozolomide and fotemustine: depletion of O6-alkyl guanine-DNA transferase in blood lymphocytes and in tumours.

BACKGROUND: The DNA repair protein O6-alkylguanine-DNA alkyl transferase (AT) mediates resistance to chloroethylnitrosoureas. Agents depleting AT such as DTIC and its new analogue temozolomide (TMZ) can reverse resistance to chloroethylnitrosoureas. We report the results of a dose finding study of TMZ in association with fotemustine. PATIENTS AND METHODS: Twenty-four patients with metastatic melanoma or recurrent glioma were treated with escalating dose of oral or intravenous TMZ ranging from 300 to 700 mg/m2, divided over two days. Fotemustine 100 mg/m2 was given intravenously on day 2, 4 hours after TMZ. AT depletion was measured in peripheral blood mononuclear cells (PBMCs) and in selected cases in melanoma metastases and was compared to TMZ pharmacokinetics. RESULTS: The maximum tolerated dose (MTD) of TMZ was 400 mg/m2 (200 mg/m2/d) when associated with fotemustine the 2nd day with myelosuppression as dose limiting toxicity. The decrease of AT level in PBMCs was progressive and reached 34% of pretreatment values on day 2. There was however wide interindividual variability. AT reduction was neither dose nor route dependent and did not appear to be related to TMZ systemic exposure (AUC). In the same patients, AT depletion in tumour did not correlate with the decrease of AT observed in PBMCs. CONCLUSIONS: PBMCs may not be used as a surrogate of tumour for AT depletion. Further study should concentrate on the pharmacokinetic pharmacodynamic relationship in tumour to provide the basis for individually tailored therapy.