A validated assay for the simultaneous quantification of six tyrosine kinase inhibitors and two active metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry.

A sensitive, sophisticated and practical bioanalytical assay for the simultaneous determination of six tyrosine kinase inhibitors (imatinib, sunitinib, nilotinib, dasatinib, pazopanib, regorafenib) and two active metabolites (N-desmethyl imatinib and N-desethyl sunitinib) was developed and validated. For the quantitative assay, a mixture of three stable isotopes as internal standards was added to human serum, standards and controls. Thereafter, samples were pre-treated using protein precipitation with methanol. The supernatant was diluted with water and injected into an ultra pressure liquid chromatographic system with an Acquity TQ tandem mass spectrometry detector. The compounds were separated on an Acquity BEH C18 analytical column (100mm×2.1mm ID, 1.7µm particle size) and eluted with a linear gradient system. The ions were detected in the multiple reaction monitoring mode. The lower limit of quantification and the linearity of all compounds generously met with the concentrations that are to be expected in clinical practice. The developed bioanalytical assay can be used for guiding TKI therapy in daily clinical practice as well as for investigator-initiated research.

Development of a nose cream containing the synthetic antimicrobial peptide P60.4Ac for eradication of methicillin-resistant Staphylococcus aureus carriage.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing problem, and current treatment options are suboptimal. Nasal carriage of MRSA is a major risk factor for infection, but nasal eradication strategies are increasingly considered to be insufficiently effective. In this study, a water-in-oil cream formulation was developed for nasal application with an antimicrobial peptide, P60.4Ac, aimed at the eradication of MRSA carriage. Quality control of the cream included the measurement of the content and release of the peptide by a validated high-performance liquid chromatography method. Stability of the peptide in the formulation was investigated including the evaluation of the effect of stress conditions. Preliminary shelf-life study of the drug formulation demonstrated that the peptide is stable in the formulation at least for 5 months. Microbial-killing assays with MRSA LUH14616 as a target demonstrated the dose-dependent antimicrobial activity of the peptide formulation.

Phenol levels during intralesional curettage and local adjuvant treatment of benign and low-grade malignant bone tumours.

BACKGROUND: Phenol is widely used for years as local adjuvant treatment for bone tumours. Despite its use for a long time, no information is available about the local concentration of phenol that is achieved in an individual patient, and the most sufficient and safe procedure to wash out the phenol after using it as local adjuvant. QUESTIONS/PURPOSES: 1. What is the initial local concentration of phenol in the tissue of the cavity wall after the application of phenol? 2. How quickly is phenol 85% diluted by washing the bone cavity with ethanol 96% solution? 3. Is the degree and speed of dilution influenced by the size of the cavity? 4. How many times should the cavity be rinsed to obtain sufficient elimination of phenol? METHODS: A basic science study was performed at respectively 16 and 10 patients, treated by intralesional curettage and adjuvant therapy for low-grade central chondrosarcoma of bone. Test 1:in 16 patients ten samples were collected of the mixture of phenol and ethanol from the bone cavity. Test 2:in ten patients, two biopsy samples were taken from the cavity wall in the bone during surgery. RESULTS: Phenol concentrations had wide variety in different patients, but all decreased by rinsing with ethanol. CONCLUSIONS: Ethanol 96% is effective to wash out local applicated phenol, by rinsing the bone cavity six times. The local concentration of phenol diminishes to an acceptable concentration of 0.2%. This study provides new insights to safely further improve the surgical technique of intralesional treatment of bone tumours.

A simplified oral flucloxacillin absorption test for patients requiring long-term treatment.

BACKGROUND: Patients with severe methicillin-sensitive Staphylococcus aureus infections are effectively treated with initial continuous intravenous (iv) flucloxacillin followed by oral maintenance therapy. As the absorption of oral flucloxacillin is variable, an oral absorption test (OAT) is used to ensure efficacious therapy. The classical OAT (test A) requires overnight fasting, interruption of iv therapy, and is laborious. We designed a simplified OAT (test B) in which iv therapy is continued and oral dosing is performed after a 1-hour fast. METHODS: In 43 hospitalized patients on iv flucloxacillin, either test A or test B was performed. In each variant, 1 g of oral flucloxacillin was given, and blood samples were taken before and at 1 and 2 hours after dosing. Flucloxacillin concentration was determined by high-performance liquid chromatography. Adequate absorption was defined as a ≥ 10 mg/L increase in flucloxacillin concentration at 1 or 2 hours after dosing. RESULTS: In a population of 43 patients (18F/25M), test A was done in 19 patients and test B in 24 patients. The groups had similar baseline characteristics such as age, renal function, gender, diagnoses, or comedication. All the patients tolerated the test without problems. The absorption was highly variable between patients. The average (SD; range) maximal increase for test A was 22.3 (11.6; 7-50) mg/L and 26.5 (12.6; 8-53) mg/L for test B. There was no significant difference between the 2 tests (P = 0.23), and 10% of the patients were poor absorbers (increase <10 mg/L). There was no influence of serum creatinine, age, or pretest flucloxacillin concentration. No clinical condition or drug use that may have impaired flucloxacillin absorption could be identified. CONCLUSIONS: We designed a simplified OAT that performs well and can be implemented easily. This test may be helpful to rationally and effectively treat patients with severe methicillin-sensitive S. aureus infections with an orally administered small-spectrum antibiotic.

Randomized trial comparing late concentration-controlled calcineurin inhibitor or mycophenolate mofetil withdrawal.

BACKGROUND: Early calcineurin inhibitor (CNI) withdrawal with mycophenolate mofetil (MMF) has not become routine practice, due to concerns about excess acute rejection. Therapeutic drug monitoring may be advantageous when the CNI or MMF is withdrawn. METHODS: This prospective, randomized, concentration-controlled withdrawal study enrolled 177 stable renal transplant recipients on maintenance CNI-based immunosuppression, combined with steroids and MMF. After the feasibility phase of the study, patients were randomized to MMF-withdrawal (target area under the time-concentration curve-cyclosporine: 3250 ng·hr/mL or tacrolimus: 120 ng·hr/mL) or CNI-withdrawal (target area under the time-concentration curve-mycophenolic acid: 75 µg·hr/mL). RESULTS: The estimated glomerular filtration rate (modification of diet in renal disease) remained significantly better after CNI elimination (59.5±2.1 mL/min vs. 51.1±2.1 mL/min, P = 0.006) up to 3 years and resulted in less functional decline, including the subgroup with an estimated glomerular filtration rate less than 50 mL/min at baseline (P = 0.03). At 6 months, one patient in the MMF-withdrawal group (1.3%) and three in the CNI-withdrawal group (3.8%) experienced acute rejection (P = 0.62). The defined higher mycophenolic acid exposure was well tolerated. CONCLUSION: These data indicate that with time the large majority of stable renal transplant recipients can be safely reduced to dual therapy with MMF or CNIs, applying concentration-controlled dosing. CNI-free patients, including those with moderate renal allograft dysfunction, have the benefit of improved renal function, whereas the risk of acute rejection after late withdrawal is low.

Mitotane has a strong and a durable inducing effect on CYP3A4 activity.

OBJECTIVE: The effects of mitotane on the pharmacokinetics (PK) of co-administered drugs are mostly unknown. The aim of the present study was to describe the effects of mitotane on the PK of the phenotypic probe midazolam and of the tyrosine kinase inhibitor sunitinib. DESIGN: A serendipitous observation was made in two of nine patients who volunteered in a sunitinib pharmacokinetic study. Both patients were diagnosed with adrenocortical carcinoma (ACC) and were exposed to mitotane. The sunitinib PK study was designed to determine the relationship between CYP3A4 activity and sunitinib exposure using 7.5 mg midazolam orally as a phenotypic probe. Patient and methods Serial blood samples for PK analysis of midazolam, 1-hydroxy-midazolam, and sunitinib were collected at steady-state sunitinib PK (between days 14 and 20). To confirm this observation in the mitotane-exposed patients, midazolam PK was evaluated in two additional patients with ACC and mitotane treatment. RESULTS: The four mitotane-treated patients showed highly induced CYP3A4 activity, even after interrupting mitotane therapy months before study entry, reflected by decreased midazolam exposure compared with the other seven patients (mean AUC(0)(-)(12 h) (95% CI): 7.6 (5.5-9.7) vs 139.0 (95.1-182.9) µg×h/l respectively P=0.001) and increased 1-hydroxy-midazolam exposure (mean AUC(0)(-)(12 h) (95% CI): 409.6 (290.5-528.7) vs 35.0 (26.4-43.6) µg×h/l, P=0.008). Sunitinib exposure was decreased in the two patients who were co-treated with mitotane (267 and 268 µg×h/l versus 1344 (1079-1609) (mean (95% CI)) µg×h/l). CONCLUSION: Mitotane has a strong and long-lasting inducing effect on CYP3A4 activity, which will result in clinically relevant interactions with multiple drugs since many drugs are metabolized by this enzyme.