A Proof-of-Concept Study of Sequential Treatment with the HDAC Inhibitor Vorinostat following BRAF and MEK Inhibitors in BRAFV600-Mutated Melanoma.

PURPOSE: The development of resistance limits the clinical benefit of BRAF and MEK inhibitors (BRAFi/MEKi) in BRAFV600-mutated melanoma. It has been shown that short-term treatment (14 days) with vorinostat was able to initiate apoptosis of resistant tumor cells. We aimed to assess the antitumor activity of sequential treatment with vorinostat following BRAFi/MEKi in patients with BRAFV600-mutated melanoma who progressed after initial response to BRAFi/MEKi. PATIENTS AND METHODS: Patients with BRAFi/MEKi-resistant BRAFV600-mutated melanoma were treated with vorinostat 360 mg once daily for 14 days followed by BRAFi/MEKi. The primary endpoint was an objective response rate of progressive lesions of at least 30% according to Response Evaluation Criteria in Solid Tumors 1.1. Secondary endpoints included progression-free survival, overall survival, safety, pharmacokinetics of vorinostat, and translational molecular analyses using ctDNA and tumor biopsies. RESULTS: Of the 26 patients with progressive BRAFi/MEKi-resistant BRAFV600-mutated melanoma receiving treatment with vorinostat, 22 patients were evaluable for response. The objective response rate was 9%, with one complete response for 31.2 months and one partial response for 14.9 months. Median progression-free survival and overall survival were 1.4 and 5.4 months, respectively. Common adverse events were fatigue (23%) and nausea (19%). ctDNA analysis showed emerging secondary mutations in NRAS and MEK in eight patients at the time of BRAFi/MEKi resistance. Elimination of these mutations by vorinostat treatment was observed in three patients. CONCLUSIONS: Intermittent treatment with vorinostat in patients with BRAFi/MEKi-resistant BRAFV600-mutated melanoma is well tolerated. Although the primary endpoint of this study was not met, durable antitumor responses were observed in a minority of patients (9%).

ELISA assay for the quantification of ipilimumab in human serum, plasma, milk, and cerebrospinal fluid.

Ipilimumab is an immune checkpoint inhibitor of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Ipilimumab has become part of the standard of care for different types of cancer. The efficacy of these treatments is limited due to immune-related toxicity and high economic costs. Dose rationalization studies based on pharmacokinetic data may help to address these limitations. For this purpose, more sensitive analytical methods are needed. We report the development and validation of the first enzyme-linked immunosorbent assay (ELISA) for sensitive determination of ipilimumab concentrations in human serum, plasma, cerebrospinal fluid (CSF), and milk. Our assay is based on the specific capture of ipilimumab by immobilized CTLA-4. The lower limit of quantifications of ipilimumab in serum, plasma, and milk are 50 ng/mL and 10 ng/mL in CSF. The ELISA method showed long-term storage stability for at least one year at -80°C and was successfully cross-validated with ultraperformance liquid chromatography coupled with tandem mass spectrometry. The ELISA method is reliable, relatively inexpensive, and can be used in serum, plasma, CSF, and milk from patients treated with ipilimumab, as evidenced by the analysis of real clinical samples.

Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of the antimalarial drug pyronaridine in human whole blood.

Malaria remains a major health concern, aggravated by emerging resistance of the parasite to existing treatments. The World Health Organization recently endorsed the use of artesunate-pyronaridine to treat uncomplicated malaria. However, there is a lack of clinical pharmacokinetic (PK) data of pyronaridine, particularly in special populations such as children and pregnant women. Existing methods for the quantification of pyronaridine in biological matrices to support PK studies exhibit several drawbacks. These include limited sensitivity, a large sample volume required, and extensive analysis time. To overcome these limitations, an ultra-performance reversed-phase liquid chromatography tandem-mass spectrometry method to determine pyronaridine was developed and validated according to international guidelines. The method enabled fast and accurate quantification of pyronaridine in whole blood across a clinically relevant concentration range of 0.500-500 ng/mL (r2 ≥ 0.9963), with a required sample volume of 50 µL. Pyronaridine was extracted from whole blood using liquid-liquid extraction, effectively eliminating the matrix effect and preventing ion enhancement or suppression. The method achieved a satisfactory reproducible sample preparation recovery of 77%, accuracy (as bias) and precision were within ±8.2% and ≤5.3%, respectively. Stability experiments demonstrated that pyronaridine was stable for up to 315 days when stored at -70°C. Adjustments to the chromatographic system substantially reduced carry-over and improved sensitivity compared to prior methods. The method was successfully applied to quantify pyronaridine in whole blood samples from a selection of pregnant malaria patients participating in the PYRAPREG clinical trial (PACTR202011812241529) in the Democratic Republic of the Congo, demonstrating its suitability to support future PK studies. Furthermore, the enhanced sensitivity allows for the determination of pyronaridine up to 42 days post-treatment initiation, enabling assessment of the terminal elimination half-life.

Impact of loperamide on the pharmacokinetics and tissue disposition of ritonavir-boosted oral docetaxel therapy; a preclinical assessment.

PURPOSE: An oral docetaxel formulation boosted by the Cytochrome P450 (CYP) 3 A inhibitor ritonavir, ModraDoc006/r, is currently under clinical investigation. Based on clinical data, the incidence of grade 1-2 diarrhea is increased with this oral docetaxel formulation compared to the conventional intravenous administration. Loperamide, a frequently used diarrhea inhibitor, could be added to the regimen as symptomatic treatment. However, loperamide is also a substrate of the CYP3A enzyme, which could result in competition between ritonavir and loperamide for this protein. Therefore, we were interested in the impact of coadministered loperamide on the pharmacokinetics of ritonavir-boosted oral docetaxel. METHODS: We administered loperamide simultaneously or with an 8-hour delay to humanized CYP3A4 mice (with expression in liver and intestine) receiving oral ritonavir and docetaxel. Concentrations of docetaxel, ritonavir, loperamide and two of its active metabolites were measured. RESULTS: The plasma exposure (AUC and Cmax) of docetaxel was not altered during loperamide treatment, nor were the ritonavir plasma pharmacokinetics. However, the hepatic and intestinal dispositions of ritonavir were somewhat changed in the simultaneous, but not 8-hour loperamide treatment groups, possibly due to loperamide-induced delayed drug absorption. The pharmacokinetics of loperamide itself did not seem to be influenced by ritonavir. CONCLUSION: These results suggest that delayed loperamide administration can be added to ritonavir-boosted oral docetaxel treatment, without affecting the overall systemic exposure of docetaxel.

A multiplex UPLC-MS/MS method for the quantification of three PD-L1 checkpoint inhibitors, atezolizumab, avelumab, and durvalumab, in human serum.

BACKGROUND AND AIM: To support pharmacokinetic studies, a multiplex UPLC-MS/MS assay was developed and validated to quantify PD-L1 checkpoint inhibitors atezolizumab, avelumab, and durvalumab in serum. METHODS: A bottom-up sample pre-treatment procedure was developed to determine atezolizumab, avelumab, and durvalumab in serum. This procedure consisted of (1) precipitation of the monoclonal antibody with ammonium sulfate, (2) reduction with dithiothreitol, (3) denaturation with methanol, and (4) tryptic digestion of the protein. The unique signature peptides resulting after sample pre-treatment of the antibodies were measured using UPLC-MS/MS with a total run time of 11 minutes. The clinical application was evaluated by analyzing 114 atezolizumab patient samples. RESULTS: The developed method was found to be accurate and precise for all three analytes over a concentration range of 3.00-150 µg/mL. No endogenous interference was present in serum samples. Cross-interference experiments showed no cross-analyte interference and acceptable cross-internal standard interference. In addition, no substantial carry-over was observed. The stable isotopically labeled signature peptides were most effective in compensating for matrix effects. Recovery based on back-calculated concentrations of calibration standards and quality control samples was found to be high. The analytes were stable for at least three freeze-thaw cycles, for 42 hours at processing conditions, for at least two days at 2-8°C in the final extract, for five days before re-injection analysis at 4°C, and long-term for at least 11 months at -70°C. The assay was tested for its applicability in clinical practice. For this purpose, 114 atezolizumab patient samples were measured. CONCLUSION: A multiplex UPLC-MS/MS assay was developed and validated to quantify atezolizumab, avelumab, and durvalumab in human serum. The applicability of this method was demonstrated by the analysis of clinical atezolizumab samples. The method is suitable to support clinical pharmacokinetic studies involving atezolizumab, avelumab, or durvalumab.

Analytical Validation of a Volumetric Absorptive Microsampling Method for Therapeutic Drug Monitoring of the Oral Targeted Anticancer Agents, Abiraterone, Alectinib, Cabozantinib, Imatinib, Olaparib, and Sunitinib, and Metabolites.

BACKGROUND: Volumetric Absorptive Microsampling (VAMS) is a useful tool for therapeutic drug monitoring (TDM) of oral targeted anticancer agents. VAMS aims to improve safety and efficacy by enabling at-home blood sample collection by patients. This study aimed to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of abiraterone, alectinib, cabozantinib, imatinib, olaparib, sunitinib, and the metabolites, Δ(4)-abiraterone (D4A), alectinib-M4, imatinib-M1, and N -desethyl sunitinib, in dried whole blood samples using VAMS to support TDM. METHODS: After the collection of 10 µL of whole blood sample using the VAMS device, the analytes were extracted from the tip using methanol with shaking, evaporated, and reconstituted in acetonitrile:0.1 mol/L ammonium hydroxide in water (1:1, vol/vol). The extracts were then analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry. Validation experiments based on the ICH M10 guideline were carried out, and stability was evaluated under shipping and storage conditions. VAMS specimens were collected in the outpatient clinic to demonstrate the applicability of the assay. RESULTS: The validated range of the method was considered accurate and precise for all analytes. Accordingly, the validation experiments met the relevant requirements, except for cross-analyte interference. Based on the stability data, shipment can be performed at room temperature within 14 days after sample collection and the VAMS specimen can be stored up to 9 months at -20 and -70°C. Samples from 59 patients were collected at the hospital. CONCLUSIONS: The developed method could be used to successfully quantify the concentrations of abiraterone, D4A, alectinib, alectinib-M4, cabozantinib, imatinib, imatinib-M1, olaparib, sunitinib, and N -desethyl sunitinib within the validated range using VAMS. Therefore, the method can be used to estimate the dried whole blood-to-plasma ratios for TDM in the clinic.

Is Higher Docetaxel Clearance in Prostate Cancer Patients Explained by Higher CYP3A? An In Vivo Phenotyping Study with Midazolam.

Patients with prostate cancer (PCa) have a lower docetaxel exposure for both intravenous (1.8-fold) and oral administration (2.4-fold) than patients with other solid cancers, which could influence efficacy and toxicity. An altered metabolism by cytochrome P450 3A (CYP3A) due to castration status might explain the observed difference in docetaxel pharmacokinetics. In this in vivo phenotyping, pharmacokinetic study, CYP3A activity defined by midazolam clearance (CL) was compared between patients with PCa and male patients with other solid tumors. All patients with solid tumors who did not use CYP3A-modulating drugs were eligible for participation. Patients received 2 mg midazolam orally and 1 mg midazolam intravenously on 2 consecutive days. Plasma concentrations were measured with a validated liquid chromatography-tandem mass spectrometry method. Genotyping was performed for CYP3A4 and CYP3A5. Nine patients were included in each group. Oral midazolam CL was 1.26-fold higher in patients with PCa compared to patients with other solid tumors (geometric mean [coefficient of variation], 94.1 [33.5%] L/h vs 74.4 [39.1%] L/h, respectively; P = .08). Intravenous midazolam CL did not significantly differ between the 2 groups (P = .93). Moreover, the metabolic ratio of midazolam to 1'-hydroxy midazolam did not differ between the 2 groups for both oral administration (P = .67) and intravenous administration (P = .26). CYP3A4 and CYP3A5 genotypes did not influence midazolam pharmacokinetics. The observed difference in docetaxel pharmacokinetics between both patient groups therefore appears to be explained neither by a difference in midazolam CL nor by a difference in metabolic conversion rate of midazolam.

Severe Immune-Related Enteritis after In Utero Exposure to Pembrolizumab.

Immune checkpoint blockade has become standard treatment for many types of cancer. Such therapy is indicated most often in patients with advanced or metastatic disease but has been increasingly used as adjuvant therapy in those with early-stage disease. Adverse events include immune-related organ inflammation resembling autoimmune diseases. We describe a case of severe immune-related gastroenterocolitis in a 4-month-old infant who presented with intractable diarrhea and failure to thrive after in utero exposure to pembrolizumab. Known causes of the symptoms were ruled out, and the diagnosis of pembrolizumab-induced immune-related gastroenterocolitis was supported by the results of histopathological assays, immunophenotyping, and analysis of the level of antibodies against programmed cell death protein 1 (PD-1). The infant's condition was successfully treated with prednisolone and infliximab.

Cortisol as Biomarker for CYP17-Inhibition is Associated with Therapy Outcome of Abiraterone Acetate.

BACKGROUND: Abiraterone acetate is an irreversible 17α-hydroxylase/C17, 20-lyase (CYP17) inhibitor approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) patients. Inhibition of this enzyme leads to low testosterone and cortisol levels in blood. There is growing evidence that clinical efficacy of abiraterone is related to the rate of suppression of serum testosterone. However, quantification of very low levels of circulating testosterone is challenging. We therefore aimed to investigate whether circulating cortisol levels could be used as a surrogate biomarker for CYP17 inhibition in patients with mCRPC treated with abiraterone acetate. PATIENTS AND METHODS: mCRPC patients treated with abiraterone acetate were included. Abiraterone and cortisol levels were measured with a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). On treatment cortisol and abiraterone concentrations were related to treatment response and progression free survival. RESULTS: In total 117 patients were included with a median cortisol concentration of 1.13 ng/ml (range: 0.03 - 82.2) and median abiraterone trough concentration (Cmin) of 10.2 ng/ml (range: 0.58 - 92.1). In the survival analyses, abiraterone Cmin ≥ 8.4 ng/mL and cortisol < 2.24 ng/mL were associated with a longer prostate-specific antigen (PSA) independent progression-free survival than patients with an abiraterone concentration ≥ 8.4 ng/mL and a cortisol concentration ≥ 2.24 ng/mL (13.8 months vs. 3.7 months). CONCLUSION: Our study shows that cortisol is not an independent predictor of abiraterone response in patients with mCRPC, but it is of added value in combination with abiraterone levels, to predict a response on abiraterone.

High accumulation of nivolumab in human breast milk: A case report.

Nivolumab is an immunotherapeutic monoclonal antibody (mAb) that is used for the treatment of several types of cancer. The evidence on its use during lactation is lacking. Here, we report on a 39-year-old woman with metastasized melanoma who was treated with 480 mg nivolumab every four weeks during lactation. Breast milk samples were collected over the course of 34 days, including two cycles of nivolumab. The highest measured concentration of nivolumab during the first cycle was 503 ng/mL at day 13. The cumulative relative infant dose (RID) over the first cycle (28 days) was 9.8 %. The highest overall measured nivolumab concentration was 519 ng/mL at day 33, five days after administration of the second nivolumab cycle. Nivolumab seems to accumulate in breast milk over two consecutive cycles, hence the RIDs of consecutive cycles are expected to be higher. To draw further conclusions regarding safety of breastfeeding during nivolumab therapy, more information about the oral bioavailability of nivolumab in newborns, the nivolumab steady-state concentrations in breast milk and its pharmacodynamic effects are needed.

Natural deletion of mouse carboxylesterases Ces1c/d/e impacts drug metabolism and metabolic syndrome development.

Mammalian carboxylesterase 1 enzymes can hydrolyze many xenobiotic chemicals and endogenous lipids. We here identified and characterized a mouse strain (FVB/NKI) in which three of the eight Ces1 genes were spontaneously deleted, removing Ces1c and Ces1e partly, and Ces1d entirely. We studied the impact of this Ces1c/d/e deficiency on drug and lipid metabolism and homeostasis. Ces1c/d/e-/- mice showed strongly impaired conversion of the anticancer prodrug irinotecan to its active metabolite SN-38 in plasma, spleen and lung. Plasma hydrolysis of the oral anticancer prodrug capecitabine to 5-DFCR was also profoundly reduced in Ces1c/d/e-/- mice. Our findings resolved previously unexplained FVB/NKI pharmacokinetic anomalies. On a medium-fat diet, Ces1c/d/e-/- female mice exhibited moderately higher body weight, mild inflammation in gonadal white adipose tissue (gWAT), and increased lipid load in brown adipose tissue (BAT). Ces1c/d/e-/- males showed more pronounced inflammation in gWAT and an increased lipid load in BAT. On a 5-week high-fat diet exposure, Ces1c/d/e deficiency predisposed to developing obesity, enlarged and fatty liver, glucose intolerance and insulin resistance, with severe inflammation in gWAT and increased lipid load in BAT. Hepatic proteomics analysis revealed that the acute phase response, involved in the dynamic cycle of immunometabolism, was activated in these Ces1c/d/e-/- mice. This may contribute to the obesity-related chronic inflammation and adverse metabolic disease in this strain. While Ces1c/d/e deficiency clearly exacerbated metabolic syndrome development, long-term (18-week) high-fat diet exposure overwhelmed many, albeit not all, observed phenotypic differences.

Assay performance and stability of uracil and dihydrouracil in clinical practice.

PURPOSE: Measurement of endogenous uracil (U) is increasingly being used as a dose-individualization method in the treatment of cancer patients with fluoropyrimidines. However, instability at room temperature (RT) and improper sample handling may cause falsely increased U levels. Therefore we aimed to study the stability of U and dihydrouracil (DHU) to ensure proper handling conditions. METHODS: Stability of U and DHU in whole blood, serum, and plasma at RT (up to 24 h) and long-term stability (≥ 7 days) at - 20 °C were studied in samples from 6 healthy individuals. U and DHU levels of patients were compared using standard serum tubes (SSTs) and rapid serum tubes (RSTs). The performance of our validated UPLC-MS/MS assay was assessed over a period of 7 months. RESULTS: U and DHU levels significantly increased at RT in whole blood and serum after blood sampling with increases of 12.7 and 47.6% after 2 h, respectively. A significant difference (p = 0.0036) in U and DHU levels in serum was found between SSTs and RSTs. U and DHU were stable at - 20 °C at least 2 months in serum and 3 weeks in plasma. Assay performance assessment fulfilled the acceptance criteria for system suitability, calibration standards, and quality controls. CONCLUSION: A maximum of 1 h at RT between sampling and processing is recommended to ensure reliable U and DHU results. Assay performance tests showed that our UPLC-MS/MS method was robust and reliable. Additionally, we provided a guideline for proper sample handling, processing and reliable quantification of U and DHU.

Carboxylesterase 1 family knockout alters drug disposition and lipid metabolism.

The mammalian carboxylesterase 1 (Ces1/CES1) family comprises several enzymes that hydrolyze many xenobiotic chemicals and endogenous lipids. To investigate the pharmacological and physiological roles of Ces1/CES1, we generated Ces1 cluster knockout (Ces1 -/- ) mice, and a hepatic human CES1 transgenic model in the Ces1 -/- background (TgCES1). Ces1 -/- mice displayed profoundly decreased conversion of the anticancer prodrug irinotecan to SN-38 in plasma and tissues. TgCES1 mice exhibited enhanced metabolism of irinotecan to SN-38 in liver and kidney. Ces1 and hCES1 activity increased irinotecan toxicity, likely by enhancing the formation of pharmacodynamically active SN-38. Ces1 -/- mice also showed markedly increased capecitabine plasma exposure, which was moderately decreased in TgCES1 mice. Ces1 -/- mice were overweight with increased adipose tissue, white adipose tissue inflammation (in males), a higher lipid load in brown adipose tissue, and impaired blood glucose tolerance (in males). These phenotypes were mostly reversed in TgCES1 mice. TgCES1 mice displayed increased triglyceride secretion from liver to plasma, together with higher triglyceride levels in the male liver. These results indicate that the carboxylesterase 1 family plays essential roles in drug and lipid metabolism and detoxification. Ces1 -/- and TgCES1 mice will provide excellent tools for further study of the in vivo functions of Ces1/CES1 enzymes.

A sensitive liquid chromatographic-mass spectrometry method for the quantification of vincristine in whole blood collected with volumetric absorptive microsampling.

Vincristine is a well-established cytotoxic drug. In paediatric populations blood collection via venipuncture is not always feasible. Volumetric absorptive microsampling (VAMS) is a less invasive method for blood collection. Furthermore, VAMS lacks the haematocrit effect on the recovery known with dried blood spots. Therefore, a liquid chromatography tandem-mass spectrometry method was developed and validated for the quantification of vincristine in whole blood collected with VAMS devices. Sample preparation consisted of solid-liquid extraction with 0.2% formic acid in water and acetonitrile. The final extract was injected on a C18 column (2.0 ×50 mm, 5 µm). Gradient elution was used and quantification was accomplished with a triple quadruple mass spectrometer operating in the positive mode. The validated concentration range was from 1 to 50 ng/mL with an intra- and inter-accuracy and precision of ± 10.3% and ≤ 7.3%, respectively. This method was able to successfully quantify vincristine concentrations in whole blood collected with VAMS from paediatric oncology patients. Vincristine concentrations in whole blood were non-linearly associated with plasma concentrations, which could be described with a saturable binding equilibrium model.

A simple extemporaneous oral suspension of aprepitant yields sufficient pharmacokinetic exposure in children.

INTRODUCTION: Aprepitant is used for the treatment of chemotherapy induced nausea and vomiting. A liquid formulation is needed for treatment of young children. However, the commercial (powder for) suspension was not available worldwide for a prolonged period of time and, therefore, a 10 mg/mL aprepitant oral suspension was extemporarily prepared to prevent suboptimal antiemetic treatment. The current pharmacokinetic study was developed to investigate whether this extemporaneous oral suspension offers an appropriate treatment option. METHODS: From 49 pediatric patients (0.7-17.9 years) 235 plasma concentrations were collected. Patients were either treated with our extemporaneous oral suspension (n = 26; 53%), commercially available capsules (n = 18; 37%), or the intravenous prodrug formulation of aprepitant (fosaprepitant, n = 5; 10%). Pharmacokinetic analyses were performed using nonlinear mixed effects modelling. RESULTS: A one-compartment model adequately described the pharmacokinetics of aprepitant in children. The bioavailability of the extemporaneous oral suspension was not significantly different to that of the capsules (P = 0.26). The observed bioavailability throughout the total population was 83% (95% CI 69%-97%). The absorption of the extemporaneous oral suspension was 39.4% (95%CI 19.5-57.4%) faster than that of capsules (mean absorption time of 1.78 h (95%CI 1.32-2.35), but was comparable to that of the commercial oral suspension. The median area under the curve after (fos)aprepitant was 22.2 mg/L*h (range 8.9-50.3 mg/L*h) on day 1. CONCLUSION: Our extemporaneous oral suspension is an adequate alternative for the commercially (un)available oral suspension in young children. An adequate exposure to aprepitant in children was yielded and the bioavailability of the extemporaneous suspension was comparable to capsules.

Bioanalytical methods for pharmacokinetic studies of antileishmanial drugs.

Bioanalytical method development and validation for the quantification of antileishmanial drugs are pivotal to support clinical trials and provide the data necessary to conduct pharmacokinetic (PK) analysis. This review provides a comprehensive overview of published validated bioanalytical assays for the quantification of antileishmanial drugs amphotericin B, miltefosine, paromomycin, pentamidine, and pentavalent antimonials in human matrices. The applicability of the assays for leishmaniasis clinical trials as well as their relevance to PK studies with emphasis on the choice of matrix, calibration range, sample volume, sample preparation, choice of internal standards, separation, and detection was discussed for each antileishmanial drug. Given that no published bioanalytical methods included multiple antileishmanial drugs in a single assay although antileishmanial shortened combination regimens currently were under investigation, it was recommended to combine various drugs in a single bioanalytical method. Furthermore, bioanalytical method development regarding target site matrix as well as applying microsampling strategies was recommended to optimize future clinical PK studies in leishmaniasis.

Development and validation of an ultra-high performance liquid chromatography coupled to tandem mass spectrometry method for the quantification of the antileishmanial drug paromomycin in human skin tissue.

Bioanalytical assay development and validation procedures were performed to quantify antiprotozoal drug paromomycin in human skin tissue by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Paromomycin, an aminoglycoside drug, is administered intra-muscularly and used in the treatment of multiple clinical presentations of the neglected tropical disease leishmaniasis. It is currently studied in the treatment of post-kala-azar dermal leishmaniasis, a disease where the Leishmania parasites divide and reside in the skin. We present a target-site bioanalytical method to accurately quantify paromomycin in human skin tissue, with the clinical purpose of quantifying paromomycin in skin biopsies from post-kala-azar dermal leishmaniasis patients originating from Sudan. Enzymatic digestion using collagenase A incubated at 37 °C overnight was employed as homogenization method to produce skin tissue homogenates. Further sample preparation was performed by protein precipitation using trichloroacetic acid and a dilution step. Final extracts were injected onto a C18 analytical column and isocratic heptafluorobutyric acid ion-pair separation and elution were employed. The chromatography system was coupled to a triple quadrupole mass spectrometer for detection. The method was validated in digestion solution over a linear range from 5 to 1000 ng/mL (r2 ≥ 0.9967) with the assay performance of accuracy and precision within acceptable criteria values as stated by the EMA guidelines. Furthermore, matrix effects were observed in human skin tissue and were corrected by the multiple deuterated paromomycin internal standard. No substantial IS-normalized matrix effect was detected along with relatively high sample preparation recovery. Consequently, digestion solution matrix serving as the preparation of calibration standards can be used as surrogate matrix for human skin tissue, which is convenient given the limited availability of control matrix. Finally, paromomycin was accurately quantified in skin of post-kala-azar dermal leishmaniasis patients originating from clinical trials in Sudan.

Overestimation of the effect of (fos)aprepitant on intravenous dexamethasone pharmacokinetics requires adaptation of the guidelines for children with chemotherapy-induced nausea and vomiting.

PURPOSE: Chemotherapy-induced nausea and vomiting (CINV) are common side effects in pediatric oncology treatment. Besides 5-HT3-antagonists, both dexamethasone and aprepitant are cornerstone drugs in controlling these side effects. Based on results of adult studies, the dexamethasone dose is reduced by 50% when combined with aprepitant, because of a drug-drug interaction, even though data on the interaction in children is lacking. The current study was developed to investigate the effect of aprepitant on dexamethasone clearance (CL) in children, in order to assess if dexamethasone dose reduction for concomitant use of aprepitant is appropriate in the current antiemetic regimen. METHODS: In total, 65 children (0.6-17.9 years), receiving intravenous or oral antiemetic therapy (dexamethasone ± aprepitant) as standard of care, were included. 305 dexamethasone plasma concentrations were determined using LC-MS/MS. An integrated dexamethasone and aprepitant pharmacokinetic model was developed using non-linear mixed effects modelling in order to investigate the effect of aprepitant administration on dexamethasone CL. RESULTS: In this population, dexamethasone CL in patients with concomitant administration of aprepitant was reduced by approximately 30% of the uninhibited CL (23.3 L/h (95% confidence interval 20.4-26.0)). This result is not consistent with the results of adult studies (50% reduction). This difference was not age dependent, but might be related to the route of administration of dexamethasone. Future studies are needed to assess the difference in oral/intravenous dexamethasone. CONCLUSION: When dexamethasone is given intravenously as a component of triple therapy to prevent CINV in children, we advise to reduce the dexamethasone dose by 30% instead of 50%.

Development and validation of a high-performance liquid chromatography tandem mass spectrometry method for the quantification of the antiparasitic and antifungal drug amphotericin B in human skin tissue.

Amphotericin B is an antifungal and antiparasitic drug used in first-line treatment of the parasitic neglected tropical disease leishmaniasis. Liposomal amphotericin B is currently studied for the treatment of cutaneous and post-kala-azar dermal leishmaniasis, where the dermis of the skin is infected with Leishmania parasites. For the optimization of known treatment regimens, accurate target-site concentrations of the drug are required. To date, no assay was available to assess human skin concentrations of amphotericin B. We here present a bioanalytical assay for the quantification of amphotericin B in 4-mm human skin biopsies. Human skin biopsies were homogenized by overnight digestion using collagenase A and were processed afterwards by simple protein precipitation using methanol. Separation and detection were achieved using a Gemini C18 column with slightly acidic chromatographic conditions and a quadrupole - linear ion trap mass spectrometer, respectively. The method was validated in digestion solution over a range of 10-2,000 ng/mL using natamycin as internal standard, with a correlation coefficient (r2) of at least 0.9974. The assay performance, accuracy and precision, were acceptable over the validated range, using international (EMA and FDA) acceptance criteria. In the skin tissue extracts, amphotericin B ion enhancement was observed, however, the internal standard (IS) corrected for this effect hence calibration standards in digestion solvent could be used as a surrogate matrix for the quantification in skin tissue. Sample preparation recoveries were low (around 27%) because of degradation of amphotericin B during digestion and sample preparation processes, albeit highly reproducible, without compromising the accuracy and precision of the method. Using this assay, amphotericin B could be detected and quantified in skin biopsies originating from treated Indian post-kala-azar dermal leishmaniasis patients.

Comparison of docetaxel pharmacokinetics between castration-resistant and hormone-sensitive metastatic prostate cancer patients.

PURPOSE: Recently, docetaxel treatment of metastatic prostate cancer patients shifted towards the hormone-sensitive stage of the disease. There are contradictive reports on differences in toxicity of docetaxel in metastatic hormone-sensitive prostate cancer (mHSPC) and metastatic castration-resistant prostate cancer (mCRPC) patients. Possible differences in toxicity might be attributed to different pharmacokinetics (PK) in the two patient populations. METHODS: Patients with mCRPC or mHSPC and a standard indication for docetaxel treatment were included in the study. All patients had suppressed serum testosterone levels (≤ 0.5 ng/mL or 1.73 nmol/L). Venous blood samples were obtained at the first docetaxel treatment, until 48 h after infusion. Plasma concentrations of docetaxel, unbound docetaxel and docetaxel metabolites were measured using validated liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) assays and compared between the two groups. Moreover, serum levels of docetaxel transporting α1-acid glycoprotein were measured and docetaxel toxicity recorded. RESULTS: A total of ten mCRPC and nine mHSPC patients were included in the study. The two cohorts differed in the number of prior treatments and opiate use, which were higher for mCRPC patients. The docetaxel PK was not different between mCRPC and mHSPC patients, with areas under the plasma concentration versus time curve (AUC0-48) 1710 [coefficient of variation (CV) 28.4%] and 1486 (CV 25.2%) ng/mL*h (p = 0.27), respectively. Also, the PK profile of unbound docetaxel, M1/M3, M2 and M4 metabolites were similar in both groups. Docetaxel doses were reduced in 50% of the mCRPC patients and 11% of the mHSPC patients. CONCLUSION: The PK profile of docetaxel was similar in mCPRC and mHSPC patients. Therefore, possible differences in toxicity between mCRPC and mHSPC patients cannot be explained by differences in docetaxel PK in our study population. These results suggest that treatment adaptations are not recommended in the new population of patients with mHSPC.