Investigating the Central Nervous System Disposition of Actinomycin D: Implementation and Evaluation of Cerebral Microdialysis and Brain Tissue Measurements Supported by UPLC-MS/MS Quantification

Actinomycin D is a potent cytotoxic drug against pediatric (and other) tumors that is thought to barely cross the blood–brain barrier. To evaluate its potential applicability for the treatment of patients with central nervous system (CNS) tumors, we established a cerebral microdialysis model in freely moving mice and investigated its CNS disposition by quantifying actinomycin D in cerebral microdialysate, brain tissue homogenate, and plasma. For this purpose, we developed and validated an ultraperformance liquid chromatography–tandem mass spectrometry assay suitable for ultra-sensitive quantification of actinomycin D in the pertinent biological matrices in micro-samples of only 20 µL, with a lower limit of quantification of 0.05 ng/mL. In parallel, we confirmed actinomycin D as a substrate of P-glycoprotein (P-gp) in in vitro experiments. Two hours after intravenous administration of 0.5 mg/kg, actinomycin D reached total brain tissue concentrations of 4.1 ± 0.7 ng/g corresponding to a brain-to-plasma ratio of 0.18 ± 0.03, while it was not detectable in intracerebral microdialysate. This tissue concentration exceeds the concentrations of actinomycin D that have been shown to be effective in in vitro experiments. Elimination of the drug from brain tissue was substantially slower than from plasma, as shown in a brain-to-plasma ratio of approximately 0.53 after 22 h. Because actinomycin D reached potentially effective concentrations in brain tissue in our experiments, the drug should be further investigated as a therapeutic agent in potentially susceptible CNS malignancies, such as ependymoma.

Relationship between plasma and intracellular concentrations of bedaquiline and its M2 metabolite in South African patients with rifampin-resistant TB

Bedaquiline is recommended for the treatment of all patients with rifampin-resistant tuberculosis (RR-TB). Bedaquiline accumulates within cells, but its intracellular pharmacokinetics have not been characterized, which may have implications for dose optimization. We developed a novel assay using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure the intracellular concentrations of bedaquiline and its primary metabolite M2 in patients with RR-TB in South Africa. Twenty-one participants were enrolled and underwent sparse sampling of plasma and peripheral blood mononuclear cells (PBMCs) at months 1, 2 and 6 of treatment and at 3 and 6 months after bedaquiline treatment completion. Intensive sampling was performed at month 2. We used non-compartmental analysis to describe plasma and intracellular exposures and a population pharmacokinetic model to explore the relationship between plasma and intracellular pharmacokinetics and the effects of key covariates. Bedaquiline concentrations from month 1 to month 6 of treatment ranged from 94.7 – 2540 ng/mL in plasma and 16.2 – 5478 ng/mL in PBMCs and concentrations of M2 over the six-month treatment period ranged from 34.3 – 496 ng/mL in plasma and 109.2 – 16764 ng/mL in PBMCs. Plasma concentrations of bedaquiline were higher than M2, but intracellular concentrations of M2 were considerably higher than bedaquiline. In the pharmacokinetic modeling, we estimated a linear increase in the intracellular-plasma accumulation ratio for bedaquiline and M2, reaching maximum effect after 2 months of treatment. The typical intracellular-plasma ratio 1 and 2 months after start of treatment was 0.61 (95%CI: 0.42-0.92) and 1.10 (95%CI: 0.74-1.63) for bedaquiline and 12.4 (95%CI: 8.8-17.8) and 22.2 (95%CI: 15.6-32.3) for M2. The intracellular-plasma ratio for both bedaquiline and M2 was decreased by 54% (95%CI: 24-72%) in HIV-positive patients compared to HIV-negative patients. Bedaquiline and M2 were detectable in PBMCs 6 months after treatment discontinuation. M2 accumulated at higher concentrations intracellularly than bedaquiline, supporting in vitro evidence that M2 is the main inducer of phospholipidosis.

Novel Glutamine Antagonist JHU395 Suppresses MYC-Driven Medulloblastoma Growth and Induces Apoptosis

Medulloblastoma is the most common malignant pediatric brain tumor. Amplification of c-MYC is a hallmark of a subset of poor-prognosis medulloblastoma. MYC upregulates glutamine metabolism across many types of cancer. We modified the naturally occurring glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) by adding 2 promoeities to increase its lipophilicity and brain penetration creating the prodrug isopropyl 6-diazo-5-oxo-2-(((phenyl (pivaloyloxy) methoxy) - carbonyl) amino) hexanoate, termed JHU395. This prodrug was shown to have a 10-fold improved CSF-to-plasma ratio and brain-to-plasma ratio relative to DON. We hypothesized that JHU395 would have superior cell penetration compared with DON and would effectively and more potently kill MYC-expressing medulloblastoma. JHU395 treatment caused decreased growth and increased apoptosis in multiple human high-MYC medulloblastoma cell lines at lower concentrations than DON. Parenteral administration of JHU395 in Nu/Nu mice led to the accumulation of micromolar concentrations of DON in brain. Treatment of mice bearing orthotopic xenografts of human MYC-amplified medulloblastoma with JHU395 increased median survival from 26 to 45 days compared with vehicle control mice (p < 0.001 by log-rank test). These data provide preclinical justification for the ongoing development and testing of brain-targeted DON prodrugs for use in medulloblastoma.

Association of Polyunsaturated Fatty Acids and Vitamin B6 with Inflammation in Adults from the National Health and Nutrition Examination Survey (NHANES) 2003–2004

Objectives Polyunsaturated fatty acids (PUFA) such as n-3 PUFA, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and n-6 PUFA, arachidonic acid (AA) may mediate inflammatory responses. Vitamin B6 deficiency has been shown to alter plasma PUFA levels. This perturbation of PUFA metabolism in vitamin B6 deficiency measured by pyridoxal-5’-phosphate (PLP) may contribute to inflammation. Thus, we aimed to examine the associations of 1) dietary EPA + DHA, and vitamin B6 with plasma ratio of AA/(EPA + DHA) by PLP level, 2) plasma AA/(EPA + DHA) and PLP with C-reactive protein (CRP), a marker for inflammation, and 3) dietary EPA + DHA, and vitamin B6 with CRP level, using NHANES. Methods NHANES datasets (2003–2004) with subjects ≥20 years were analyzed, accounting for survey design and sample weights (n = 4486). The significance level was P < 0.05. Covariates were age, gender, ethnicity, BMI, smoking, alcohol, total energy, dietary supplements, physical activity, and NSAIDs, depending on analyses. Multiple linear regression assessed the association of dietary EPA + DHA, and vitamin B6 with plasma ratio of AA/(EPA + DHA) by PLP level (Low: < 20 nmol/L, High: ≥20 nmol/L). Next, multivariate logistic regression predicted the associations of plasma AA/(EPA + DHA) and PLP with CRP level (Low: ≤3 mg/L, Moderate to High: > 3 mg/L); then, dietary EPA + DHA, and B6 with CRP level. Results In the low PLP level, dietary EPA + DHA was negatively associated with plasma ratio of AA/(EPA + DHA) (B = ─5.29, SE = 0.84, P = < .0001), but B6 intake was not, whereas, in the high PLP level, both dietary EPA + DHA (B = ─2.99, SE = 0.53, P = < .0001) and dietary vitamin B6 (B = ─0.21, SE = 0.04, P = 0.0001) were inversely associated with plasma AA/(EPA + DHA). Further, low PLP level was associated with greater odds of moderate to high CRP level compared to high PLP level (adjusted OR (aOR): 2.8, 95% CI: 1.93–4.04, P = < .0001), but plasma AA/(EPA + DHA) was not. In addition, both dietary EPA + DHA (aOR: 0.5, 95% CI: 0.23–0.98, P = 0.04) and vitamin B6 (aOR: 0.8, 95% CI: 0.68–0.95, P = 0.009) were inversely associated with moderate to high CRP level. Conclusions Our findings show that low plasma PLP level and low vitamin B6 intake are associated with inflammation, and the relationship may be through their effect on PUFA metabolism, suggesting that increased intake of vitamin B6 and EPA and DHA may protect against inflammation. Funding Sources N/A.

Population Pharmacokinetics/Pharmacodynamics of Dabrafenib Plus Trametinib in Patients with BRAF-Mutated Metastatic Melanoma

Patients treated with dabrafenib/trametinib (DAB/TRA) exhibit a large interindividual variability in clinical outcomes. The aims of this study were to characterize the pharmacokinetics of DAB, hydroxy-dabrafenib (OHD), and TRA in BRAF-mutated patients and to investigate the exposure–response relationship for toxicity and efficacy in metastatic melanoma (MM) patients. Univariate Fisher and Wilcoxon models including drug systemic exposure (area under the plasma concentration curve, AUC) were used to identify prognostic factors for the onset of dose-limiting toxicities (DLT), and Cox models for overall (OS) and progression-free survival (PFS). Seventy-three BRAF-mutated patients were included in pharmacokinetic (n = 424, NONMEM) and 52 in pharmacokinetic/pharmacodynamic analyses. Age and sex were identified as determinants of DAB and OHD clearances (p < 0.01). MM patients experiencing DLT were overexposed to DAB compared to patients without DLT (AUC: 9624 vs. 7485 ng∙h/mL, respectively, p < 0.01). Eastern Cooperative Oncology Group Performance Status (ECOG PS) ≥ 2 and plasma ratio AUCOHD/AUCDAB ≥ 1 were independently associated with shorter OS (HR: 6.58 (1.29–33.56); p = 0.023 and 10.61 (2.34–48.15), p = 0.022, respectively). A number of metastatic sites ≥3 and cerebral metastases were associated with shorter PFS (HR = 3.25 (1.11–9.50); p = 0.032 and HR = 1.23 (1.35–10.39), p = 0.011; respectively). TRA plasma exposure was neither associated with toxicity nor efficacy. Our results suggest that early drug monitoring could be helpful to prevent the onset of DLT in MM patients, especially in fragile patients such as the elderly. Regarding efficacy, the clinical benefit to monitor plasma ratio AUCOHD/AUCDAB deserves more investigation in a larger cohort of MM patients.