Antifungal serum concentration monitoring: an update.

Invasive fungal infections (IFIs) are occurring with increasing incidence and are associated with significant morbidity and mortality. Understanding the relationship between the pharmacokinetic and pharmacodynamic properties of antifungals is essential to optimize the potential for favourable clinical and microbiological outcomes while minimizing risks of treatment-related toxicity. Antifungal serum concentrations may aid in the determination of appropriate dosing in select circumstances. The polyene and echinocandin classes of antifungals lack sufficient data to justify serum concentration monitoring in routine clinical practice. In contrast, serum concentration monitoring of flucytosine may help to reduce the risk of treatment-related haematological toxicity. Determination of itraconazole serum concentrations is advised in situations where the drug is used for prolonged periods to treat serious IFIs (such as invasive aspergillosis or histoplasmosis) because of variability in absorption following oral administration (most notable for the capsule formulation). The use of serum concentration monitoring during therapy with the extended-spectrum triazoles (i.e. voriconazole and posaconazole) is still evolving, due primarily to inter-patient variability in drug exposure combined with sparse data regarding relationships with efficacy (posaconazole) and both safety and efficacy (voriconazole).

Vertical-flow paper SERS system for therapeutic drug monitoring of flucytosine in serum.

A number of life-saving drugs require therapeutic drug monitoring (TDM) for safe and effective use. Currently, however, TDM is performed using sophisticated analytical techniques relegated to central labs, increasing the cost per test and time to answer. Here, using a novel vertical flow membrane system with inkjet-printed surface enhanced Raman sensors, along with a portable spectrometer, we demonstrate a low cost and easy to use device to quantify levels of flucytosine, an antifungal that requires TDM for effective patient care, from undiluted human serum. To our knowledge, this work represents the first report of a passive vertical flow sample cleanup method with surface enhanced Raman detection. We first investigated and optimized the parameters of the vertical flow system for the detection of flucytosine in spiked serum samples. Then, using an optimized vertical-flow system utilizing nitrocellulose membranes and a paper SERS sensor, we achieved detection of down to 10 µg mL-1 flucytosine in undiluted serum, with quantitative detection across the entire therapeutic range. This system reduces the assay time to about 15 min, far quicker than the current gold standards. We anticipate that this novel system will enable near-patient therapeutic drug monitoring, leading to the safe and effective administration of a number of life-saving drugs. Furthermore, it will spawn the development of SERS detection systems capable of separating target analytes from real-world biological matrices.

Utilizing online-dual-SPE-LC with HRMS for the simultaneous quantification of amphotericin B, fluconazole, and fluorocytosine in human plasma and cerebrospinal fluid.

Amphotericin B (AMB), fluconazole (FZ), and fluorocytosine (FC) are recommended for HIV-associated cryptococcal meningitis (CM) patients as preferred antibiotics. This study presents a fast and automated online-dual-solid phase extraction (SPE)-LC coupled with high resolution mass spectrometer (HRMS) method to simultaneously measure the concentrations of AMB, FZ, and FC in human plasma and cerebrospinal fluid (CSF). Automated sample clean-up was performed on the human plasma and CSF samples with stop-flow heart-cutting two dimensional (2D) separation using a online-dual-SPE system, allowing retention and accumulation of AMB, FZ, and carbamazepine (CBZ, Internal standard (IS)) by the Oasis®HLB cartridge, and retention and accumulation of FC and 5-methylcytosine hydrochloride (MC, IS) by the HyperSep Hypercarb cartridge respectively. Followed by LC elution, quantification by Q-Exactive Hybrid Quadrupole-Orbitrap with targeted-selected ion monitoring (t-SIM) mode was applied to simultaneously determine the concentrations of AMB, FZ and FC. The bioanalysis was achieved in a total running time of 7min. The method was fully validated according to FDA guidelines. The lowest limit of quantification (LLOQ) was 0.04, 0.04, and 0.40µgmL-1 for AMB, FZ, and FC, respectively. AMB, FZ, and FC levels were linear in the ranges of 0.04-2.00µgmL-1, 0.04-2.00µgmL-1 and 0.40-20.00µgmL-1, respectively. The method showed good performance for human plasma and CSF samples with linearity (R2>0.99), intra-day and inter-day precision (relative standard deviation, RSD<4.32% and <4.06%, respectively), recovery (89.93-93.28% and 90.09-93.58%, respectively) and matrix effect (96.35-103.78% and 92.32-101.48%, respectively). The validated method was successfully applied in real samples of Chinese patients. Overall, our results indicate that this fully automated, sensitive, and reliable online-dual-SPE-LC-HRMS method is effective for therapeutic drug monitoring (TDM) of AMB, FZ, and FC levels.

Flucytosine-miconazole treatment of Candida peritonitis. Its use during continuous ambulatory peritoneal dialysis.

A case of Candida parapsilosis peritonitis occurred in a patient undergoing continuous ambulatory peritoneal dialysis (CAPD). Treatment with oral doses of flucytosine and intravenous doses of miconazole was effective. No interruption in CAPD was necessary, and acceptable flucytosine levels were obtained in the serum and dialysate. This case and information available in the literature suggest that Candida peritonitis can be treated effectively with antimicrobial agents that are well tolerated by the patient, and removal of the dialysis catheter is not always necessary for cure.

Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients.

We performed a pilot trial in refractory cancer patients to investigate the feasibility of intratumoral injection of TAPET-CD, an attenuated Salmonella bacterium expressing the E. coli cytosine deaminase gene. A total of three patients received three dose levels of TAPET-CD (3 x 10(6)-3 x 10(7) CFU/m(2)) via intratumoral injection once every 28 days as long as progression of disease or intolerable toxicity was not observed. From days 4 to 14 of each 28 day cycle, patients also received 5-fluorocytosine (5-FC) at a dose of 100 mg/kg/day p.o. divided three times daily. Six cycles of treatment were administered. No significant adverse events clearly attributable to TAPET-CD were demonstrated. Two patients had intratumor evidence of bacterial colonization with TAPET-CD, which persisted for at least 15 days after initial injection. Conversion of 5-FC to 5-fluorouracil (5-FU) as a result of cytosine deaminase expression was demonstrated in these two patients. The tumor to plasma ratio of 5-FU for these two colonized patients was 3.0, demonstrating significantly increased levels of 5-FU at the site of TAPET-CD colonization and insignificant systemic spread of the bacteria. In contrast, the tumor to plasma ratio of 5-FU of the patient who did not show colonization of TAPET-CD was less than 1.0. These results support the principle that a Salmonella bacterium can be utilized as a delivery vehicle of the cytosine deaminase gene to malignant tissue and that the delivered gene is functional (i.e. able to convert 5-FC to 5-FU) at doses at or below 3 x 10(7) CFU/m(2).

Assessment of antifungal therapy in an 800-gram infant with candidal arthritis and osteomyelitis.

A 720-g premature newborn developed disseminated candidiasis during treatment with systemic antibiotics and total parenteral nutrition through an umbilical arterial catheter. Clinical features were typical for candidal skeletal infection at this age and included warmth and fusiform swelling of the lower extremities together with radiographic evidence of osteolysis and cortical bone erosion. Candida albicans was cultured from blood, urine, joint fluid, and a bone aspirate. The infection was cured with a 44-day course of amphotericin B and flucytosine (5-fluorocytosine). Antifungal therapy was monitored closely with serum drug levels and laboratory tests for bone marrow toxicity and renal dysfunction. Serum levels of both drugs were comparable to those achieved in older patients treated with similar doses. Significant concentrations of amphotericin B were detected in serum four and 17 days after completion of therapy, indicating a slow rate of elimination similar to that which occurs in adults. There was no evidence of drug-induced toxicity other than transient elevation in the fractional urinary excretion of sodium. This suggests that antifungal therapy may be effectively and safely administered to infants in dose schedules similar to those used for older patients.

Ocular penetration of 5-fluorocytosine.

Ocular penetration of 5-fluorocytosine (5FC) was studied in uninfected rabbits after subconjunctival and oral administration. With oral administration, 5FC achieved therapeutic levels in both the vitreous and aqueous humors. The use of a pharmacokinetic model permitted objective comparison of kinetic events within the eye chambers and the serum. The rates of entry and elimination in the vitreous were found to be slower than those in the aqueous, but the mean concentration over 24 hr was the same. The therapeutic levels achieved in the aqueous after subconjunctival administration were of shorter duration, and no detectable levels occurred in the vitreous. Oral administration is clearly therapeutically superior to subconjunctival administration in this model.

Enzyme rate assay for 5-fluorocytosine.

Modification of an enzyme assay for 5-fluorocytosine, from an end point to a rate analysis, has resulted in a method that can provide results within 10 minutes. The modification also rendered the assay less susceptible to interference from bilirubin and triglycerides. This obviated the need to correct 5-fluorocytosine results for icteric or moderately lipemic samples. The performance history of the enzyme rate method has shown that it is reliable, quick, cost-effective, and suitable for use in a clinical laboratory.

Falsely elevated serum creatinine levels secondary to the presence of 5-fluorocytosine.

The Kodak Ektachem (Eastman Kodak Co., Rochester, NY) is a new clinical chemistry analyzer that uses an enzymatic method to measure creatinine. The authors report the case of a patient with falsely elevated creatinine levels that were caused by the presence of 5-fluorocytosine. A review of the literature confirmed that this can occur, but well documented reports are not found. In order to determine the magnitude of this interference, the authors plotted creatinine levels versus 5-FC concentration. Significant interference is seen with therapeutic levels of 5-FC. Both clinicians and pathologists should be aware of this phenomenon.

The measurement of serum 5-fluorocytosine levels in the presence of miconazole.

Miconazole is effective against a wide variety of fungi, and may be of value when used in combination with 5-fluorocytosine in the treatment of life-threatening mycotic infections. Because the hematologic toxicity of 5-fluorocytosine is related to high serum levels, the effects of miconazole on the standard disc diffusion assay for 5-fluorocytosine were studied. When standards were made in either undiluted or 10% pooled human serum, no difference in zone size between 5-fluorocytosine alone and the combination of 5-fluorocytosine and miconazole was found. It is concluded that, in this assay system, miconazole does not affect the disc diffusion test for serum 5-fluorocytosine levels.

Model appropriateness and population pharmacokinetic modeling.

The purpose of this study was to define model appropriateness, identifying the individual elements thereof, and to set out a framework within which model appropriateness could be determined for population pharmacokinetic (PPK) models. Model appropriateness was defined by stating the problem to be solved, with the intended use of the model being the pivotal event. The elements of model appropriateness were identified with the type of model (descriptive vs. predictive) determining which elements of model appropriateness need to be executed. An example is presented to show how model appropriateness is determined for the optimal application of PPK models. It was determined that PPK models are developed to solve problems. Model appropriateness depends on identifying the problem, as well as stating the intended use of the model, and requires evaluation of the model for goodness of fit, reliability, and stability if intended for descriptive purposes; for predictive models, validation would be an additional requirement. Descriptive models are used to explain variability in the pharmacokinetics (PK) of a drug, while predictive models are developed to extrapolate beyond the immediate study population. For those models used for predictive purposes, strong assumptions are made about the relationship to the underlying population from which the data were collected. As an example of determining model appropriateness, a PPK model for 5-fluorocytosine was developed, using NONMEM, version IV. The model was evaluated and validated by the process of percentile bootstrapping. From the PPK model, the range of expected serum concentrations based on two widely used dosing methods (Sanford and the University of California at San Diego [UCSD]) was simulated (Pharsight Trial Designer software). These results indicated that the UCSD method performed well and has the advantage of recommending convenient dosing intervals. In conclusion, considering and applying the principles of model appropriateness to PPK models will result in models that can be applied for their intended use with confidence. Model appropriateness was efficiently established and determined to address the problem of comparing competing dosing strategies.

Rapid fluorimetric determination of 5-fluorocytosine in serum.

A rapid fluorimetric procedure for the determination of the fungicide 5-fluorocytosine in serum is described. The method is high in precision (S.D. 1.6 mug/ml at 50 mug/ml) and specificity and can be performed in 60-90 minutes. The results obtained for samples from six patients showed excellent agreement with those obtained by traditional microbiological methods.

Use of in vitro release tests for the prediction of the in vivo behavior and the development of flucytosine controlled-release capsules.

The antifungal substance flucytosine (5-fluorocytosine) was chosen as a model drug for studying the development of a controlled-release capsule dosage form based mainly on in vitro drug release tests. A solution and various capsule formulations of flucytosine were orally administered to beagle dogs, and the resulting plasma concentration-time profiles were determined. Special attention was directed to multiple-unit hard-gelatin capsules that exhibited a controlled-release effect in vitro, but not in vivo. The respective plasma profile was treated by the technique of numerical deconvolution to obtain the corresponding in vivo release profile (i.e., the drug release from the dosage form in the gastrointestinal tract of the dog). The influence of different experimental conditions of the USP paddle method on the release profile was investigated. By adding surfactant to the dissolution medium and omission of the sinker device, which is used to prevent the capsule from floating, the in vitro release rate markedly increased and correlated well with the in vivo release. Based on this adjusted in vitro test, the formulation was modified to achieve a more pronounced controlled-release effect. The plasma profile resulting from the reformulated multiple-unit capsules was in good agreement with the predictions made by numerical convolution of the in vitro release profile.