Oxidative stress plays a major role in tissue damage under pathological conditions and has
been targeted for pharmacological prevention of nerve cell injury in a number of
neurological diseases including stroke, Alzheimer’s disease (AD), amyotrophic lateral
sclerosis (ALS). Unfortunately, although necessary for the development of new drugs, useful
diagnostic biomarkers of oxidative stress for neurological diseases are not yet available. In
the first part of the current study, we demonstrated that protein alterations in cerebral spinal
fluid (CSF) of stroke patients, including expression and post-translational modifications such
as oxidation, were investigated using two-dimensional electrophoresis (2-DE). Protein
oxidation was detected with immunoblotting using 2, 4-dinitrophenylhydrazine (2, 4-DNPH)
and anti-DNP antibody. In the CSF proteins of stroke patients, approximately 48 spots were
altered after the onset of stroke, including alpha-1-acid glycoprotein, alpha-1-microglobulin,
vitamin D-binding protein precursor (DBP), beta-actin, transthyretin, and complement C4. In
addition, human serum albumin (HSA) was a major oxidized protein found after stroke in
CSF. Albumin is well known as an endogenous anti-oxidant in the blood. We suggest that
albumin provided protection against reactive oxygen species, preventing neuronal cell death
in the CA1 region following 10 min of transient forebrain ischemia. We also found that the
function of albumin as an ROS scavenger could be changed by modification of its oxidation
status in vivo. A second issue that also needs consideration regarding development of new
CNS drugs concerns the ability of various compounds to cross the blood-brain barrier (BBB).
In several clinical trials, antioxidant drugs such as vitamin E have shown no beneficial effect
because an effective therapeutic concentration could not be achieved in the brain. The BBB
penetration rate is intimately associated with success of CNS targeted drug development.
Pharmacokinetics and pharmacodynamics studies on the target organ are very important in the process of drug development. In second part of this study, we studied the
pharmacokinetics and pharmacodynamics of a new drug candidate, AAD-2004, which has
been proposed as having antioxidant properties capable of targeting neurodegenerative
diseases. LC-MS was first used to validate pharmacokinetics and pharmacodynamics
analysis of AAD-2004. This compound reduced oxidative stress products such as
nitrotyrosine and 8-OHdG in ALS mice. Moreover, it showed maximal effects at Cmax = ~
2.12 μg/ml and AUC = ~3.92 μg.hr/ml in blood that could realistically be expected to
achieve more than effective dose in the brain.
