Devin F. Welty

Knockout of Glutamate Transporters Reveals a Major Role for Astroglial Transport in Excitotoxicity and Clearance of Glutamate

Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

A summary of mechanistic hypotheses of gabapentin pharmacology

Although the cellular mechanisms of pharmacological actions of gabapentin (Neurontin) remain incompletely described, several hypotheses have been proposed. It is possible that different mechanisms account for anticonvulsant, antinociceptive, anxiolytic and neuroprotective activity in animal models. Gabapentin is an amino acid, with a mechanism that differs from those of other anticonvulsant drugs such as phenytoin, carbamazepine or valproate. Radiotracer studies with [14C]gabapentin suggest that gabapentin is rapidly accessible to brain cell cytosol. Several hypotheses of cellular mechanisms have been proposed to explain the pharmacology of gabapentin: 1. Gabapentin crosses several membrane barriers in the body via a specific amino acid transporter (system L) and competes with leucine, isoleucine, valine and phenylalanine for transport. 2. Gabapentin increases the concentration and probably the rate of synthesis of GABA in brain, which may enhance non-vesicular GABA release during seizures. 3. Gabapentin binds with high affinity to a novel binding site in brain tissues that is associated with an auxiliary subunit of voltage-sensitive Ca2+ channels. Recent electrophysiology results suggest that gabapentin may modulate certain types of Ca2+ current. 4. Gabapentin reduces the release of several monoamine neurotransmitters. 5. Electrophysiology suggests that gabapentin inhibits voltage-activated Na+ channels, but other results contradict these findings. 6. Gabapentin increases serotonin concentrations in human whole blood, which may be relevant to neurobehavioral actions. 7. Gabapentin prevents neuronal death in several models including those designed to mimic amyotrophic lateral sclerosis (ALS). This may occur by inhibition of glutamate synthesis by branched-chain amino acid aminotransferase (BCAA-t).

Effects of anticonvulsant drug gabapentin on the enzymes in metabolic pathways of glutamate and GABA

Gabapentin is a novel anticonvulsant drug. The anticonvulsant mechanism of gabapentin is not known. Based on the amino acid structure of gabapentin we explored its possible effects on glutamate and gamma-aminobutyric acid (GABA) metabolism in brain as they may relate to its anticonvulsant mechanisms of action. Gabapentin was tested for its effects on seven enzymes in the metabolic pathways of these two neurotransmitters: alanine aminotransferase (AL-T), aspartate aminotransferase (AS-T), GABA aminotransferase (GABA-T), branched-chain amino acid aminotransferase (BCAA-T), glutamine synthetase (Gln-S), glutaminase (GLNase), and glutamate dehydrogenase (GDH). In the presence of 10 mM gabapentin, only GABA-T, BCAA-T, and GDH activities were affected by this drug. Inhibition of GABA-T by gabapentin was weak (33%). The Ki values for inhibition of cytosolic and mitochondrial forms of GABA-T (17-20 mM) were much higher than the Km values for GABA (1.5-1.9 mM). It is, therefore, unlikely that inhibition of GABA-T by gabapentin is clinically relevant. As with leucine, gabapentin stimulated GDH activity. The GDH activity in rat brain synaptosomes was activated 6-fold and 3.4-fold, respectively, at saturating concentrations (10 mM) of leucine and gabapentin. The half-maximal stimulation by gabapentin was observed at approximately 1.5 mM. Gabapentin is not a substrate of BCAA-T, but it exhibited a potent competitive inhibition of both cytosolic and mitochondrial forms of brain BCAA-T. Inhibition of BCAA-T by this drug was reversible. The Ki values (0.8-1.4 mM) for inhibition of transamination by gabapentin were close to the apparent Km values for the branched-chain amino acids (BCAA) L-leucine, L-isoleucine, and L-valine (0.6-1.2 mM), suggesting that gabapentin may significantly reduce synthesis of glutamate from BCAA in brain by acting on BCAA-T.

Gabapentin anticonvulsant action in rats: disequilibrium with peak drug concentrations in plasma and brain microdialysate

The concentration-time profile of [14C]gabapentin (GBP) in plasma and brain interstitial fluid (ISF) was determined following a single 15 mg/kg intravenous bolus dose to rats. Brain ISF was sampled with a microdialysis probe in striatum. Blood was also collected serially to 4 h postdose. At termination, brain was sectioned into regions and [14C]GBP concentrations were determined. Anticonvulsant effects were determined by maximal electroshock in rats with identical dosing. Plasma [14C]GBP declined linearly after dosing while brain ISF [14C]GBP concentration peaked at approximately 1 h and then declined in parallel with plasma concentration. Throughout, brain ISF [14C]GBP concentration was approximately 3-6% of [14C]GBP concentration in plasma. However, at 4 h postdose, whole brain tissue [14C]GBP concentration was equal to or greater than the concentration of [14C]GBP in plasma. Maximal anticonvulsant effect lagged behind both plasma and brain ISF GBP concentrations. The anticonvulsant effect of GBP is delayed by time-dependent events other than distribution from blood to brain.

Potential Treatment of Amyotrophic Lateral Sclerosis with Gabapentin: A Hypothesis

Objective: To provide the biochemical rationale for the use of the new anticonvulsant agent gabapentin as a treatment for amyotrophic lateral sclerosis (ALS). Background: ALS is a neuropathologic disorder of the central nervous system characterized by a progressive loss of upper and lower motor neurons. Although the etiopathology of ALS is incompletely known, it is hypothesized that glutamatergic neurotransmission is related to neuropathology. Glutamate is an excitatory amino acid neurotransmitter that is cytotoxic when overexpressed at synaptic terminals, probably through a calcium-related mechanism. The concentration of glutamate in cerebrospinal fluid is increased in patients with ALS. The increased extracellular concentrations of glutamate may be caused by a decreased capacity of glutamate transport in brain tissue and/or abnormal glutamate metabolism. Recent success with the glutamate release inhibitor riluzole in well-controlled clinical trials supports the excitotoxic mechanism of neuropathology in patients with ALS. Potential Treatment For Als: Gabapentin has demonstrated neuroprotective properties in a model of chronic glutamate toxicity in vitro. Although the neuroprotective mechanism of action of gabapentin is currently unknown, it is hypothesized here that gabapentin decreases the rate of formation of glutamate derived from the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine. The proposed decrease in formation of glutamate from BCAAs may decrease the pool of releasable glutamate and therefore compensate for diminished glutamate uptake capacity and/or abnormal glutamate metabolism in patients with ALS. Conclusions: Based on this rationale, it is proposed that gabapentin may provide a beneficial effect in the treatment of patients with ALS.

The Temporal Effect of Food on Tacrine Bioavailability

A four‐way cross‐over study was performed to assess the temporal effect of food on the rate and extent of tacrine (Cognex, THA) absorption after drug administration to healthy, older volunteers. Each volunteer received four single 40‐mg THA doses at 1‐week intervals. Doses were administered after an 8‐hour overnight fast, 1 hour before a standard breakfast, 15 minutes after beginning a standard breakfast, and 2 hours after completion of a standard breakfast. Gastrointestinal side effects were most frequently reported after drug administration to fasted subjects. Mean Cmax and AUC(0–∞) values after THA administration during breakfast (9.9 ng/mL and 70.2 ng · hr/mL) and 2 hours after breakfast (11.6 ng/mL and 74.2 ng · hour−1 · mL−1) were significantly lower than values determined after administration of THA to fasting subjects (15.8 ng/mL, and 91.8 ng · hour−1 · mL−1). Little effect was evident when THA was administered 1 hour before breakfast.

Neuropharmacokinetics of two investigational compounds in rats: Divergent temporal profiles in the brain and cerebrospinal fluid

Two investigational compounds (FRM-1, (R)-7-fluoro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide and FRM-2, (R)-7-cyano-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide) resided in rat brain longer than in systemic circulation. In Caco-2 directional transport studies, they both showed good intrinsic passive permeability but differed significantly in efflux susceptibility (efflux ratio of <2 and ∼7, respectively), largely attributed to P-glycoprotein (P-gp). Capitalizing on these interesting properties, we investigated how cerebrospinal fluid (CSF) concentration (CCSF) would be shaped by unbound plasma concentration (Cu,p) and unbound brain concentration (Cu,b) in disequilibrium conditions and at steady state. Following subcutaneous administration, FRM-1CCSF largely followed Cu,p initially and leveled between Cu,p and Cu,b. However, it gradually approached Cu,b and became lower than, but parallel to Cu,b at the terminal phase. In contrast, FRM-2CCSF temporal profile mostly paralleled the Cu,p but was at a much lower level. Upon intravenous infusion to steady state, FRM-1CCSF and Cu,b were similar, accounting for 61% and 69% of the Cu,p, indicating a case of largely passive diffusion-governed brain penetration where CCSF served as a good surrogate for Cu,b. On the contrary, FRM-2CCSF and Cu,b were remarkably lower than Cu,p (17% and 8% of Cu,p, respectively), suggesting that FRM-2 brain penetration was severely impaired by P-gp-mediated efflux and CCSF underestimated this impact. A semi-physiologically based pharmacokinetic (PBPK) model was constructed that adequately described the temporal profiles of the compounds in the plasma, brain and CSF. Our work provided some insight into the relative importance of blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) in modulating CCSF.

Hepatic microsomal induction profile of carbamic acid [[2,6-bis(1- methylethyl)phenoxy] sulfonyl]-2,6-bis(1-methylethyl) phenyl ester, monosodium salt (PD138142-15), a novel lipid regulating agent.

Induction of hepatic microsomal cytochrome P450 produced by carbamic acid [2,6-bis(1-methylethyl)phenoxy]sulfonyl]-2,6-bis(1-methylethyl) phenyl ester, monosodium salt (PD138142-15), a novel water-soluble inhibitor of acyl-CoA: cholesterol acyltransferase, was examined in male and female rats, dogs, and monkeys, and in male guinea pigs. Relative to control, PD138142-15 increased hepatic microsomal total spectral P450 in all species examined. Hepatic microsomal ethoxyresorufin-O-deethylase, pentoxyresorufin-O-dealkylase, and peroxisomal carnitine acetyltransferase activities and cyanide-insensitive Beta-oxidation were affected only marginally. Erythromycin-N-demethylase activity was increased (2- to 6-fold) in all three species in which it was examined (rat, dog and pig). Marked increases in immunoreactive P450 3A were noted in the rats and dogs, while slight increases were seen in monkeys. Pharmacokinetic studies of PD138142-15 in rats and dogs revealed pronounced decreases (80-90%) in plasma Cmax and AUC within 2 weeks of initiation of daily dosing. In spite of the marked decline in plasma drug levels, efficacy in dogs, as determined by serum cholesterol levels, was maintained for up to 6 weeks with continued dosing. Potential acid (gastric) breakdown products of PD 138142-15 were examined for their hepatic cytochrome P450 induction profiles in rats adn were found to differ both quantitatively and qualitatively from profiles produced by the parent compound. This suggested that induction observed in rats was due to parent PD138142-15 and not to any of the known potential acid breakdown products. The cumulative data establish that PD 138142-15 is an inducer of P450 3A in rats and dogs. The results also suggest that P450 3A is induced in monkeys and pigs as well, although the data are less definitive. Decreases in plasma drug levels imply that the compound may be an autoinducer in dogs and rats. The maintenance of efficacy in spite of decreased drugs levels in dogs suggests that the effects on serum cholesterol are due to a metabolite or that cholesterol lowering effects occur before the compound is metabolized by the liver.