A number of new drugs for neurodegenerative and inflammatory disease focus on target sites primarily within the brain. With human ED50 at mg or lower, the concentrations for assessments of these drugs in zebrafish models have been at nanogram or picogram scale.
While certain neuro drugs have excellent therapeutic effect, they also have narrow therapeutic range making translational dosing an extremely sensitive subject.
To ensure we are reading drug concentrations with high accuracy levels, a three step, fool proof method has been identified.
Step 1: Ensure brain sections are from site of drug activity.
For drugs acting on mid brain region, only the mid brain sections were collected as opposed to using the whole brain tissue. Tissue collection was normalized with weight of the collected slice and the protein concentration using biochemistry.
Step 2: Applications of innovation tissue extraction methods
Solvents to dissolve brain tissue were normalized such that minimal physical disruption would be needed, enabling a low table top life time of the sample. Following cell lysis, sample purification was done by innovative solid phase extraction procedure leading to high quality tissue extract with maximum recovery rates.
Step 3: Immediate read outs were performed without sample storage process leading to a high quality sample input. Regression analysis generated highly sensitive limits of detection within 2 hours to 6 hours post dosing at 2 hours interval. Group variation was minimal due to the use of graded animals for each treatment allowing to detect picogram levels of drugs dosed to the animals. Excretion profiles were obtained from the swim water of the larvae.
Clonazepam, a benzodiazepine, detected from a single larvae dose, 2 hours prior; Brain tissue clonazepam directly corelated with levels of unbound clonazepam in the blood.
Conclusion: A highly sensitive method to measure brain penetrating drugs have been made possible by the applications of new chemistry and lab processes. This finds applications with small molecules, hybrid molecules, degraders and small oligonucleotide therapies.
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