Asymmetric synthesis and pharmacology of methylphenidate and its para-substituted derivatives.

We report the first asymmetric synthesis of the individual enantiomers of methylphenidate (1). From d-pipecolic acid, the (2R,2'R) and (2S,2'R) enantiomers of 1 were obtained in > 99% optical purity while the (2S,2'S) and (2R,2'S) enantiomers of 1 were derived from l-pipecolic acid in 96% optical purity. The versatility of this methodology is demonstrated with the synthesis of the (2R,2'R) and (2S,2'S) enantiomers of p-bromo and p-methoxy derivatives in similar yields and enantiomeric purities. Comparative neurochemical assessments of these synthesized enantiomers at purported dopamine, norepinephrine, and serotonin uptake sites along with locomotor activity studies in rats are also reported.

Comparison of the superfused efflux of preaccumulated D-[3H]aspartate and endogenous L-aspartate and L-glutamate from rat cerebrocortical minislices.

Because the validity of the use of preaccumulated isotopic excitatory amino acids (EAAs) to index the depolarization-evoked release of endogenous EAAs has been questioned, we compared the K(+)-evoked efflux of preaccumulated D-[3H]aspartate from rat cerebrocortical minislices with that of endogenous L-aspartate and L-glutamate. Release of all EAAs increased with the rate of superfusion. Using the most rapid rate (1.6 ml/min), transient elevations in [K+] caused a concentration-dependent increase, with 50 mM K+ evoking a 33-, 23- and 93-fold increase in the efflux of D-[3H]aspartate, L-aspartate and L-glutamate, respectively; this efflux was Ca(2+)-dependent and tetrodotoxin insensitive. Under polarized conditions (5 mM K+), 1 mM kainic acid increased the efflux of preaccumulated and endogenous EAAs. These elevations were not blocked by the competitive kainate receptor antagonist 6,7-dinitroquinoxaline-2-3-dione (DNQX) and were not affected by removing Ca2+ ions. We conclude that in superfused cortical minislices, the efflux of preaccumulated D-[3H]aspartate provides a robust and reliable index of the release of endogenous L-aspartate and L-glutamate.

The release and uptake of excitatory amino acids in rat brain: effect of aging and oxidative stress.

The excitatory amino acids (EAAs) L-aspartate and L-glutamate constitute the major neurotransmitters in the mammalian brain. This study established the influence of aging and oxidative stress on the release and uptake of EAAs. The high affinity uptake of D-[3H]aspartate in synaptosomal fractions of the neostriatum, hippocampus, and neocortex was not significantly different in Fisher 344/Norwegian Brown hybrid rats aged 3, 12, 24, and 37 months. Similarly, the K(+)-evoked efflux of endogenous aspartate and glutamate from neocortical minislices was also unaffected by age. To examine the possibility that EAA nerve terminals become more vulnerable to oxidative stress with age, the influence of an inhibitor of the electron transport chain (sodium cyanide) on EAA uptake and release was determined. Although cyanide inhibited D-[3H]aspartate uptake and potentiated the potassium-evoked efflux of aspartate and glutamate in a Ca(2+)-independent fashion, neither of these changes were influenced by age. Thus, the functional integrity of EAA nerve terminals and their vulnerability to oxidative stress are both preserved in normal aging. The potency of cyanide to inhibit D-[3H]aspartate uptake did, however, display regional variability: hippocampus > neocortex > neostriatum (IC50 = 1.2 +/- 0.2 mM, 1.9 +/- 0.1 mM and 2.7 +/- 0.2 mM, respectively), suggesting that EAA nerve terminals in the hippocampus may be selectively vulnerable to oxidative stress.

Activities and kinetic properties of lumbar cerebrospinal fluid cholinesterases in relation to clinical diagnosis, severity, and progression of Alzheimer's disease.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities of lumbar cerebrospinal fluid (CSF) have been measured in seventeen patients with a clinical diagnosis of probable Alzheimer's disease (Prob AD), possible Alzheimer's disease (Poss AD), or dementia of non-Alzheimer aetiology (Non-AD). The three diagnostic groups did not differ with regard to the Km or saturation kinetic properties of AChE and BChE. The CSF AChE activity was significantly higher in Prob AD than in Non-AD patients. The groups did not differ significantly in BChE activity. The ratio of AChE to BChE activity was significantly higher in both the Prob AD and Poss AD groups than in the Non-AD group, and the ranges of values in the Prob AD and Non-AD groups did not overlap. Among patients in the Prob AD group, severity of dementia was correlated with both AChE activity and the AChE/BChE ratio, and progression of dementia over time was also correlated with AChE/BChE. The AChE/BChE ratio correlated more strongly than AChE with severity and progression of dementia in Prob AD patients, and also better distinguished them from Non-AD patients, suggesting that AChE/BChE may be the more useful marker for diagnosis of AD. It is not clear from the results whether AChE/BChE is useful for diagnosis of the complex dementia cases in the Poss AD group.

The ratio of acetylcholinesterase to butyrylcholinesterase influences the specificity of assays for each enzyme in human brain.

A single-vial extraction radiometric assay was used to measure acetylcholinesterase and butyrylcholinesterase in human post-mortem brain tissue. Data are presented regarding the differential inhibition of acetylcholinesterase and butyrylcholinesterase in several brain areas from a case of Alzheimer's disease and a case without neuropsychiatric disease. The results indicate that neither enzyme is entirely specific for its substrate, and the necessity for using inhibitors depends upon the ratio of acetylcholinesterase to butyrylcholinesterase in a particular sample.