Gennethel Pennick

Development and Validation of a High-Performance Liquid Chromatography Assay for Voriconazole

ABSTRACT An analytical method for the determination of voriconazole (UK-109,496; Pfizer) in plasma was developed and validated. The method utilizes solid-phase extraction technology and high-performance liquid chromatography. The lower limit of quantitation is 0.2 μg/ml, and the range of linearity tested was 0.2 to 10 μg/ml.

Comparison of High-Performance Liquid Chromatographic and Microbiological Methods for Determination of Voriconazole Levels in Plasma

ABSTRACT A new selective high-performance liquid chromatography (HPLC) method with UV detection for the determination of the investigational triazole voriconazole in human plasma by using acetonitrile precipitation followed by reverse-phase HPLC on a C18column was compared with a simple agar well diffusion bioassay method with Candida kefyr ATCC 46764 as the assay organism. Pooled plasma was used to prepare standard and control samples for both methods. The results of analyses with spiked serum samples (run as unknowns) were concordant by the bioassay and HPLC methods, with expected values being obtained. HPLC demonstrated an improved precision (3.47 versus 12.12%) and accuracy (0.81 versus 1.28%) compared to those of the bioassay method. The range of linearity obtained by both methods (from 0.2 to 10 μg/ml for HPLC and from 0.25 to 20 μg/ml for the bioassay) includes the range of concentrations of voriconazole (from 1.2 to 4.7 μg/ml) which are considered clinically relevant. Although either methodology could be used for the monitoring of patient therapy, the smaller variability observed with HPLC compared to that observed with the bioassay favors the use of HPLC for pharmacokinetic studies.

Posaconazole Therapeutic Drug Monitoring: a Reference Laboratory Experience

We read with great interest the review by Andes et al. (1) describing antifungal therapeutic drug monitoring (TDM). We agree that there are limited data suggesting serum posaconazole concentrations predict the efficacy of prophylaxis or treatment with this agent. Prior studies have reported only mean serum drug levels by patient group (those with breakthrough invasive fungal infections versus those who remained uninfected) or by quartile with an average response rate to therapy (2, 5, 7-9). Serum posaconazole levels have also been repeatedly shown to have a large degree of interpatient variability (4-7). However, it is our experience that undetectable posaconazole levels are commonly encountered despite attempts to maximize absorption. One of two early posaconazole prophylaxis studies reported mean serum posaconazole levels of 1.470 μg/ml in patients with chronic graft-versus-host disease (GVHD) and 0.958 μg/ml in those with acute GVHD (8) However, the average posaconazole levels were 0.611 μg/ml in patients who developed breakthrough infection and 0.922 μg/ml in those who remained uninfected (5). The other and concurrently published prophylaxis study reported a mean serum posaconazole level of 0.583 ± 381 μg/ml—a mean value below the average of those who experienced breakthrough infection in the aforementioned GVHD prophylaxis study (2). A study evaluating posaconazole as salvage therapy for invasive aspergillosis correlated therapeutic response to serum drug concentrations by quartiles and found those with a mean serum drug level of 0.134 μg/ml had only a 24% response rate (9). Additionally, the FDA briefing document recommends a goal posaconazole average serum drug concentration of >0.700 μg/ml (3). We retrospectively reviewed 202 consecutive serum posaconazole concentrations obtained between 26 December 2007 and 30 December 2008 by The Fungus Testing Laboratory, San Antonio, TX, a reference laboratory specializing in fungal identification, susceptibility testing, and antifungal drug concentrations. Drug levels were obtained with a validated high-performance liquid chromatography assay (range, 0.125 to 5.0 μg/ml [equipment manufactured by Beckman Coulter, Fullerton, CA]). Levels less than 0.125 μg/ml were reported as undetectable, and levels greater than 5.0 μg/ml were diluted 1:2, reextracted, and reanalyzed. Our review confirmed the infrequent obtainment of levels suggestive of efficacy as described above. In our series, 158/202 (78.2%) posaconazole levels were <0.92 μg/ml and 135/202 (66.8%) were <0.611 μg/ml, a value that may represent patients at increased risk of breakthrough infection while receiving posaconazole prophylaxis. Although the exact value known to be therapeutic in the treatment of invasive mycoses has not been determined, it is noteworthy that 33/202 (16.3%) in our series were undetectable (<0.125), 35/202 (17.3%) were <0.134 μg/ml (the value associated with only a 24% response rate in posaconazole use as salvage therapy for invasive aspergillosis), and 142/202 (70.3%) were <0.700 μg/ml (the value cited by the FDA to place patients at increased risk) (Fig. ​(Fig.11). FIG. 1. Distribution of serum posaconazole levels obtained by the Fungus Testing Laboratory, San Antonio, TX, from 26 December 2007 through 30 December 2008. When drug concentrations thought to be subtherapeutic are observed, attempts at maximizing drug availability/absorption should be undertaken. Strategies proven to maximize posaconazole exposure include administration with or after a high-fat meal, with any meal or nutritional supplement, with an acidic beverage, or in divided doses and with the avoidance of acid suppression drugs (6). However, it is common for low serum posaconzole levels to be found despite these maneuvers, and these differences observed between serum drug levels within clinical trials and “real-world” use may have an important clinical impact on the frequency of TDM and choice of antifungal agent. We thus recommend repeat testing of serum posaconazole levels after the above attempts to maximize absorption. Although previous reports have questioned the need for TDM with posaconazole pending outcome studies based on levels, our data suggest that testing should be performed to ensure that measurable drug is present and therefore has the potential for efficacy.

Serial Plasma Voriconazole Concentrations after Allogeneic Hematopoietic Stem Cell Transplantation

ABSTRACT Plasma voriconazole concentrations vary considerably between patients receiving standard dosing, and trough voriconazole concentrations are known to affect efficacy and toxicity. Temporal variations in serial plasma voriconazole concentrations through the course of therapy in hematopoietic stem cell transplantation patients has not been carefully described. Paired voriconazole concentrations in 64 patients were studied to determine the predictability of the second concentration based on the first. The difference between the two values was ≤5% in six patients. In 25 patients, the second concentration was higher by a median of 40%. In 33 patients, the subsequent concentration was lower by a median of 59%. For patients with an initial concentration of <2 μg/ml, the correlation between the two values was poor (r = 0.24; P < 0.17). For those with an initial concentration of ≥2 μg/ml, the correlation was good (r = 0.72; P < 0.0001). There was no relationship between the magnitude of the change and the time elapsing between the two measurements. Among the 43 patients who had an initial concentration of ≥1 μg/ml, the two voriconazole measurements were strongly correlated (r = 0.66, P < 0.0001), but only 67% had a voriconazole serum concentration of ≥1 μg/ml on the second measurement. No studied variables were reliable predictors in identifying concentrations above or below 1 or 2 μg/ml. Our data suggest that variations in voriconazole concentrations are unpredictable despite standard dosing, and the acceptability of a concentration on one occasion cannot be extrapolated to future concentrations in the same patient. This suggests that ongoing therapeutic drug monitoring and dose adjustment may be beneficial in patients requiring prolonged voriconazole therapy.

A Reference Laboratory Experience of Clinically Achievable Voriconazole, Posaconazole, and Itraconazole Concentrations within the Bloodstream and Cerebral Spinal Fluid

ABSTRACT Interest in antifungal therapeutic-drug monitoring has increased due to studies demonstrating associations between concentrations and outcomes. We reviewed the antifungal drug concentration database at our institution to gain a better understanding of achievable triazole drug levels. Antifungal concentrations were measured by high-performance liquid chromatography (HPLC), ultraperformance liquid chromatography and single-quadrupole mass spectrometry (UPLC/MS), or a bioassay. For this study, only confirmed human bloodstream (serum or plasma) and cerebral spinal fluid (CSF) concentrations of voriconazole, posaconazole, and itraconazole were analyzed. The largest numbers of bloodstream and CSF samples were found for voriconazole (14,370 and 173, respectively). Voriconazole bloodstream concentrations within the range of 1 to 5.5 μg/ml represented 50.6% of samples. Levels below the lower limit of quantification (0.2 μg/ml) were observed in 14.6% of samples, and 10.4% of samples had levels of ≥5.5 μg/ml. CSF voriconazole levels ranged from undetectable to 15.3 μg/ml and were <0.2 μg/ml in 11% of samples. Posaconazole bloodstream concentrations were ≥0.7 and ≥1.25 μg/ml in 41.6% and 18.9% of samples, respectively. Posaconazole was detected in only 4 of 22 CSF samples (undetectable to 0.56 μg/ml). Itraconazole levels, as measured by UPLC/MS, were ≥0.5 μg/ml in 43.3% and were undetectable in 33.9% of bloodstream samples. In contrast, when measured by a bioassay, itraconazole/hydroxyitraconazole bloodstream concentrations were ≥1.0 μg/ml in 72.9% of samples and were undetectable in 18% of samples. These results indicate that there is marked variability in bloodstream concentrations achieved with these three azoles. In addition, many levels within the bloodstream for each azole and for voriconazole and posaconazole in the CSF were undetectable or below thresholds associated with efficacy.

STEADY-STATE PLASMA CONCENTRATIONS OF ITRACONAZOLE AFTER ORAL ADMINISTRATION IN KEMP'S RIDLEY SEA TURTLES, LEPIDOCHELYS KEMPI

Abstract Pharmacokinetic studies of antifungal agents in reptiles are uncommon. Itraconazole, which has been used prophylactically in juvenile sea turtles suffering from hypothermia (cold stunning) on a regular basis, was evaluated for steady-state plasma concentrations. Five Kemps ridley sea turtles (Lepidochelys kempi) receiving itraconazole at several dosages in a rehabilitation program had blood collected within 24 hr to estimate dosing frequency. Subsequently, serial blood samples of Kemps ridley sea turtles that were given itraconazole at several dosages for 30 days to treat cold stunning were collected at various intervals to evaluate itraconazole plasma concentrations. Tissue samples were collected from one Kemps ridley that died during rehabilitation. Plasma concentrations of itraconazole (and of hydroxyitraconazole [OH-ITRA], one of its major bioactive metabolites) were determined using a modified, validated reverse-phase high-performance liquid chromatography technique. Itraconazole concentrations in tissues were determined by bioassay to be far greater than the plasma concentrations measured in any of the turtles. At a 15-mg/kg dosage, the half-life (t1/2) was 75 hr for itraconazole and 55 hr for OH-ITRA. All dosages produced adequate concentrations in some turtles, but consistent therapeutic concentrations were produced only at 15 mg/kg q72hr and 5 mg/kg s.i.d., with the latter producing the highest plasma concentrations.

Accelerated Metabolism of Voriconazole and Its Partial Reversal by Cimetidine

ABSTRACT We report a case of accelerated metabolism of voriconazole during therapy for invasive pulmonary aspergillosis, resulting in subtherapeutic levels. Target voriconazole levels were restored with high dosages of voriconazole (up to 40 mg/kg of body weight/day) and the addition of cimetidine as a cytochrome P450 enzyme inhibitor.

Micafungin Concentrations from Brain Tissue and Pancreatic Pseudocyst Fluid

ABSTRACT We report the attainment of micafungin concentrations from brain tissue and pancreatic pseudocyst fluid from two patients with invasive candidiasis. Micafungin was present in low levels at both body sites, indicating limited penetration into central nervous system (CNS) tissue and pancreatic fluid. Further studies are needed to fully characterize its pharmacokinetics at these locations, as micafungin may potentially serve as an alternative antifungal therapy for CNS or pancreatic candidal infections for which the currently recommended first-line therapy fails.

Pharmacokinetics of Anidulafungin in Pleural Fluid during the Treatment of a Patient with Candida Empyema

ABSTRACT Candida empyema is a serious complication of disseminated candidiasis. However, little is known about the intrapleural pharmacokinetics of echinocandins. We report the penetration of anidulafungin into the pleural fluid of a patient with Candida tropicalis empyema. The anidulafungin ratio for the area under the concentration-time curve from 0 h to the last measurement between pleural fluid and serum values was only 0.125 (12.5%), with pleural fluid concentrations ranging between 0.67 and 0.88 μg/ml.

Pharmacokinetics of Liposomal Amphotericin B in Pleural Fluid

ABSTRACT We report the penetration of liposomal amphotericin B into the pleural fluid of a patient with pulmonary zygomycosis and empyema. The ratio of area under the concentration-versus-time curve in pleural fluid (AUCpleural fluid) to that in serum (AUCserum) for liposomal amphotericin B over 24 h was 9.4%, with pleural fluid concentrations of 2.12 to 4.91 μg/ml. Given the relatively low level of intrapleural penetration of liposomal amphotericin B, chest tube drainage may be warranted for successful treatment of zygomycotic empyema.