Topiramate [TPM, 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate] (RWJ-17021-000, formerly McN-4853) is a structurally novel antiepileptic drug (AED). The preclinical anticonvulsant profile suggests that TPM acts primarily by blocking the spread of seizures. TPM was highly effective in the maximal electroshock (MES) seizure test in rats and mice. Activity was evident < or = 0.5 h after oral administration and lasted at least 16 h. The ED50 values 4 h after oral dosing were 13.5 and 40.9 mg/kg in rats and mice, respectively. TPM blocked pentylenetetrazol (PTZ)-induced clonic seizures at high doses in mice (ED50 = 1,030 mg/kg orally, p.o.). With motor incoordination and loss of righting reflex used as indicators of neurologic impairment, the neuroprotective index (TD50/MES ED50) for TPM was equivalent or superior to that of several approved AEDs. In mice pretreated with SKF-525A (a P450 enzyme inhibitor), the anticonvulsant potency was either increased or unaffected when TPM was tested 0.5, 1, or 2 h after i.p. administration, suggesting that TPM rather than a metabolite was the active agent. In mice pretreated with reserpine or tetrabenazine, the activity of TPM in the MES test was markedly reduced. TPM was inactive in a variety of receptor binding, neurotransmitter uptake, and ion channel tests. TPM weakly inhibited erythrocyte carbonic anhydrase (CA) activity. However, the anticonvulsant activity of TPM appears to differ mechanistically from that of acetazolamide.
Summary: Purpose: This study investigated the effectiveness of topiramate (TPM) as an inhibitor of six isozymes of carbonic anhydrase (CA). Methods: The inhibition constants (Ki) of TPM and acetazolamide (AZM) for CA I, CA II, CA III, CA IV, CA V, and CA VI were determined for human (HCA), rat (RCA), or mouse (MCA). The activity of CA was studied by using purified isozymes, erythrocytes, subcellular fractions of kidney or brain, and saliva, and was assayed at 37°C or 25°C by 18O mass spectrometry and/or by measuring the pH shift at 0°C. Results: Topiramate Ki values for HCA I, HCA II, HCA IV, and HCA VI were ∼100, 7, 10, and >100 μM, respectively. TPM Ki values for RCA I, RCA II, RCA III, RCA IV, and RCA V were ∼180, 0·1 to 1, >100, 0·2 to 10 and 18 μM, respectively. For RCA II and RCA IV, the Ki values were temperature dependent. TPM Ki values for MCA II and MCA IV ranged between 1 and 20 μM. Conclusions: These results indicate that TPM is more potent as an inhibitor of CA II and CA IV than of CA I, CA III, and CA VI. In all three species, AZM was usually 10 to 100 times more potent than TPM as an inhibitor of CA isozymes.
The role of carbonic anhydrase in de novo lipid synthesis was examined by measuring [1-14C]acetate incorporation into total lipids, fatty acids and non-saponifiable lipids in freshly isolated rat hepatocytes. Two carbonic anhydrase inhibitors, trifluoromethylsulphonamide (TFMS) and ethoxozolamide (ETZ) decreased incorporation of 14C into total lipids. Both fatty acid and non-saponifiable lipid components of the total lipid were inhibited to approximately the same extent by 100 microM TFMS (29 +/- 0.3% and 35 +/- 0.3% of control respectively in replicate studies). However, neither drug significantly affected ATP concentrations or the transport activity of Na+/K(+)-ATPase, two measures of cell viability. To establish the site of this inhibition, water-soluble 14C-labelled metabolites from perchloric acid extracts of the radiolabelled cells were separated by ion-exchange chromatography. TFMS inhibited 14C incorporation into citrate, malate, alpha-oxoglutarate and fumarate, but had no effect on incorporation of 14C into acetoacetate. Since ATP citrate-lyase, the cytosolic enzyme that catalyses the conversion of citrate into acetyl-CoA, catalyses an early rate-limiting step in fatty acid synthesis, levels of cytosolic citrate may be rate controlling for de novo fatty acid and sterol synthesis. Indeed citrate concentrations were significantly reduced to 37 +/- 6% of control in hepatocytes incubated with 100 microM TFMS for 30 min. TFMS also inhibited the incorporation of 14C from [1-14C]pyruvate into malate, citrate and glutamate, but not into lactate. This supports the hypothesis that TFMS inhibits pyruvate carboxylation, i.e. since all of the 14C from [1-14C]pyruvate converted into citric acid cycle intermediates must come via pyruvate carboxylase (i.e. rather than pyruvate dehydrogenase). Our findings indicate a role for carbonic anhydrase in hepatic de novo lipogenesis at the level of pyruvate carboxylation.
We have assayed carbonic anhydrase activity (carbonate dehydratase, carbonate hydro-lyase, EC 4.2.1.1) and icarbonate permeability in suspensions of broken and intact guinea pig mitochondria by monitoring the disappearance of C16'080. We found significant activity in preparations from liver and skeletal muscle, but not in preparations from heart muscle, brain, and kidney. Intact mitochondria containing carbonic anhydrase produce a two-phase acceleration of the disappearance of the labeled CO2, which indicates that the enzyme is located in a region more accessible to CO2 than to HCOj-. Acetazolamide inhibits the enzyme activity instantly in broken mitochondria but only after a delay in intact mitochondria, indicating that the enzyme is in a region not immediately accessible to the inhibitor. Sonication of mitochondria containing carbonic anhydrase activity releases the enzyme, which remains in the supernatant after sedimentation of the submitochondrial particles. This shows that mitochondrial carbonic anhydrase is in the matrix compartment and not in, or bound to, the inner membrane. The activity of the enzyme increases markedly with increasing pH. The enzyme activity of intact mitochondria is greater than that of the broken mitochondria at the same pH of the suspending fluid, corresponding to an intramitochondrial pH that is 0.2-0.5 unit more alkaline.CO2 plays an important role in mitochondrial metabolism. The enzymes of the tricarboxylic acid cycle that produce CO2 (1) are located within the mitochondrial matrix (2), as are those enzymes that fix CO2 in the pathways of gluconeogenesis and urea production (3, 4). Prior to 1972, the few studies of the interaction of CO2 and HCO% with mitochondria dealt with the permeability of the inner mitochondrial membrane to these two species and with the possible existence of a mitochondrial carbonic anhydrase. Chappell and Crofts (5) showed that the inner mitochondrial membrane was essentially impermeable to HC0% but readily permeable to C02, a finding now widely accepted (6). Early reports of mitochondrial carbonic anhydrase (7-9) were cautious because of the problem of contamination, with the exception of that of Karler and Woodbury (10). Rossi (11) found 4% of the carbonic anhydrase of rat liver homogenate in the mitochondrial fraction and showed that it was an intramitochondrial enzyme. Mitochondria incubated with 10 ,gM acetazolamide showed no activity. Holton (12) found carbonic anhydrase activity in isolated rat liver mitochondria with both penetrant CO2 and nonpenetrant HCO as substrates. In contrast to these two positive findings with liver mitochondria, Deprez and Francois (13) found no carbonic anhydrase activity in mitochondrial preparations from various tissues, including liver. In all the above studies, carbonic anhydrase activity was measured by change in pH.A functional role for C02/HC0% in providing a counteranion for energy-linked mitochondrial Ca2+ uptake was proposed by Elder (14). Elder and Lehninger (15,16) showed that HCO3 as such cannot se...
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