Development of online high speed capillary electrophoresis fluorescence assays for 3’, 5’-cyclic guanosine monophosphate (cGMP) and pharmacokinetic monitoring of intranasal administration of drugs to the mammalian brain

Abstract

An innovative analytical system coupling online high speed microdialysis (MD) to capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection has been developed for in vivo monitoring of chemical dynamics in the mammalian brain. Previously used for simultaneous in vivo monitoring and analysis of amino acid neurotransmitters; my research applied this powerful technique to the development of novel fluorescence assays for 1) the detection and monitoring of the intracellular messenger 3’, 5’ cyclic Guanosine Monophosphate (cGMP) and 2) monitoring pharmacokinetics of intranasal administration of drugs to the rat brain. cGMP plays a significant regulatory role in numerous biological processes, including modulation of protein phosphorylation. Of specific interest is cGMP’s proposed significance in the glutamate nitric oxide (NO) signaling pathway. As a downstream indicator of NO activity, the study of cGMP may lead to a greater understanding of the steps involved in reduction of oxidative stress and excitotoxic effects associated with human diseases and neurologically related disorders. Development of the cGMP assay was performed using a custom self built online, high speed MD-CE-LIF instrument. Derivatization was carried out in an online reaction with the guanine selective fluorogenic reagent phenylglyoxal (PGO). A flow gated interface was used to load the sample onto the separation capillary. Fluorescence was excited using the 364 nm line of an argon-ion laser in a sheath flow detector cell. Emission was collected at 90o, passed through a band-pass filter, and collected on a photomultiplier tube. Optimization and characterization of the cGMP assay was carried out to achieve maximum sensitivity, selectivity, resolution, and speed of analysis. Online detection limits of 14-19 nM (sample collected through an in-house manufactured microdialysis probe) have been achieved. cGMP was resolved at nanomolar concentrations with a temporal resolution on the order of 25-50 seconds. In vivo characterization of cGMP was performed in the rat striatum. Application of pharmacological agents via reverse microdialysis was performed to induce the stimulation of cGMP production. Experiments were carried out with high-K+ aCSF, glutamate, phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), and nitric oxide donor 2-(N-N-Diethylamino)-diazenolate-2-oxide (DEANO). Studies to establish the pharmacology of cGMP are done for two reasons: 1) confirm the identity of the cGMP peak, and 2) test current hypotheses regarding cGMP’s function in vivo. Working in concert with the Alzheimer’s Research Center at Regions Hospital in St. Paul, we developed a novel fluorescence assay for the detection and monitoring of fluorescein in the rat brain following intranasal (i.n.) administration. A significant challenge in the development of clinical therapies for central nervous system (CNS) diseases and injuries is difficulty in delivering the agents directly to the target CNS regions due to blood brain and blood-cerebrospinal fluid barriers. The precise mechanisms underlying nose to brain transport post i.n. administration are not completely understood. MD-CE-LIF analysis of i.n. administered fluorescent compounds may elucidate possible pathways and mechanisms underlying transport from the nasal passages to the CNS and aid clinical development of drug therapies capable of directly targeting CNS regions associated with the pathology of neurological damage and neurodegenerative diseases. Development of the fluorescein assay was performed using the custom built online high speed MD-CE-LIF instrument. A flow gated interface was used to load the sample onto the separation capillary. Fluorescence was excited using a 488 nm solid state laser in a sheath flow detector cell. Emission was collected at 90o, passed through a band-pass filter, and collected on a photomultiplier tube. Optimization and characterization of the fluorescein assay was carried out to achieve maximum sensitivity, selectivity, resolution, and speed of analysis. The developed fluorescein assay achieved an online (sampled through an in house manufactured microdialysis probe) limit of detection (LOD) of 34 ± 4 pM with a temporal resolution on the order of 50 seconds. Electrophoretic separation of fluorescein was complete in 17-23 seconds at 23 kV. Animal protocol development and in vivo animal handling was carried out to characterize the fluorescein assay in vivo post intranasal administration of fluorescein. Fluorescein has been detected in the rat brain at picomolar to nanomolar concentrations and the first high temporal resolution pharmacokinetic profile of fluorescein in the rat olfactory bulb was achieved. The optimized fluorescein assay was used to study the effectiveness of chitosan and hydroxypropyl-β-cyclodextrin (HPβCD) as nasal absorption/nasal mucosal membrane permeability enhancers of intransally delivered fluorescein with brain pharmacokinetics measured in the rat olfactory bulb via MD-CE-LIF. Whole blood samples were drawn and analyzed via microdialysis to elucidate fluorescein’s likely nose to brain delivery pathways. Pharmacokinetic profiles of fluorescein in the olfactory bulb were attained with a temporal resolution not previously demonstrated or published in any intranasal drug delivery study. Initial chitosan experiments did not indicate increased absorption and detection of fluorescein in the rat olfactory bulb. HPβCD demonstrated significant fluorescein enhancement in the brain relative to intransally administered fluorescein without an absorption enhancer. The temporal resolution advantages associated with MD-CE-LIF allowed information to be measured on a time scale not possible in previous cGMP assays, and provided the first high temporal pharmacokinetic profile of fluorescein in the rat olfactory bulb. A high potential is indicated for application of MD-CE-LIF to brain pharmacokinetic study in the development of future intranasal drug therapies directly targeting the central nervous system.