Development of an in vitro drug recovery model for paediatric tuberculous meningitis using microdialysis with in vivo application

Lourens, Rentia

Development of an in vitro drug recovery model for paediatric tuberculous meningitis using microdialysis with in vivo application

Master Thesis

2022

Abstract

Introduction: Tuberculous Meningitis (TBM) is the most devastating form of tuberculosis, leading to high rates of death and disability. Yet little has changed in drug regimens to treat TBM, in part due to the limitations of studying drugs at the site of the disease: the vast majority of studied samples are from spinal cerebrospinal fluid (CSF), most sampling is sporadic, and drug concentrations are usually reported as total (bound and unbound) concentrations. However, spinal CSF may not be the same as CSF derived directly from the brain (ventricular CSF), continuous or semi-continuous sampling would provide better pharmacokinetic data, and the unbound fraction of the drug would be more informative because it is the pharmacologically active component. Microdialysis (MD) is a method that is used in advanced clinical care to measure molecules in tissue, including the brain. It has some limitations, however, including the difficulty of calculating the relative recovery and potential anatomical compartmental effects in the brain. We aimed to adapt the method to address these limitations and for the first time produce high frequency data of unbound drugs in ventricular CSF using rifampicin in TBM as a model in vitro and in vivo. Aims: We aimed to establish a novel technique, using MD to continually measure Rifampicin in ventricular CSF of TBM patients. This involved 1) developing an appropriate system that could be used clinically, 2) determining the proportion of the drug that is recovered via the MD catheter (termed the recovery), 3) identifying parameters that affects the recovery, and 4) applying the model to the clinical setting. Methods: We developed a system with an embedded MD catheter to continuously drain artificial CSF with known concentration of Rifampicin and protein. The MD catheter had 20 kDa semipermeable membrane that allowed passage of only unbound drugs. To examine recovery, we compared rifampicin concentrations in hourly samples through the MD catheter (termed microdialysate) to total rifampicin in control CSF samples (obtained from the system before passage through the catheter). These were analysed using liquid chromatography – tandem mass spectrometry (LC-MS/MS). We considered several factors that may affect recovery in clinical practice, running several iterations that varied rifampicin concentration, protein concentration, MD perfusion pump rates, CSF draining rates, and system pressure (to mimic intracranial pressure). The method was then applied to paediatric TBM patients, where the MD catheter was placed in-line with an extra-ventricular drain (EVD) and the factors identified in vitro were stabilised. Rifampicin concentrations were measured in hourly microdialysate samples over 24 hours, and compared to control samples of ventricular CSF using LCMS/MS. Results: For the in vitro experiments, the effect of several variables on relative recovery were identified. Increased recovery was seen with a higher CSF draining rate and increased rifampicin concentration, while decreased recovery was seen with increased MD perfusion rate, increased protein concentration, and unexpectedly, increased pressure in the system (to mimic intracranial pressure). When translated to two patients with TBM we aimed to maintain intracranial pressure within a narrow range, and stable CSF drainage and MD pump rates. The in vivo unbound rifampicin concentrations from microdialysate were used to draw a 24-hour pharmacokinetic curve. The peak concentration (Cmax) in microdialysate was 221 ng/mL before enzyme induction (where drug administration had just started) and 100 ng/mL after enzyme induction (where the patient had been on Rifampicin for more than two weeks). The time of the peak (Tmax) in microdialysate was 11 hours after drug administration. The ratio of the unbound drug concentration to the total drug concentration of the control samples varied. Conclusion: This novel method is feasible to study unbound drug concentrations directly and continuously in the brain and provides high quality data for advanced neuropharmacokinetics. Factors in the clinical setting can be accounted for, and microdialysate results can be compared against sporadic control sampling. Clinical translation of this method was successful. These early data suggest very low concentrations of rifampicin in the brain with standard care. The results raise interesting hypotheses about the binding of Rifampicin to protein in the human brain. Our method can be applied to other drugs and data obtained from the method can inform dosing requirements and improve future drug development.

Keywords

Neurosurgery

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