RESUMO
We have compared the cellular responses to simvastatin (Simva) and atorvastatin (Atorva), two potent HMG-CoA reductase inhibitors. The two drugs exhibited similar IC50's for inhibition of either rat or human reductase, and single oral dosing in rats showed the compounds to be nearly equipotent at inhibiting hepatic cholesterol synthesis. Treatment of rats with Simva or Atorva in the feed for four days yielded comparable inductions of hepatic reductase activity and reductase protein. For example, 0.05% Simva induced reductase activity 27.3 +/- 9.1 fold and 0.05% Atorva induced activity 26.9 +/- 4.7 fold. This adaptive response was also studied in HepG2 cells, a human hepatoblastoma line, cultured for 24 h in delipidated serum and then for an additional 24 h with Simva or Atorva. Over a broad range (10 nM-10 microM), both drugs caused similar inductions of reductase activity, reductase protein, and reductase mRNA. Under all conditions, the drugs induced similar changes in the ratio of mRNA/protein suggesting that Simva and Atorva have similar effects on both transcriptional and post-transcriptional regulatory machinery. Moreover, reductase in cells treated with Simva or Atorva for 22 h responded similarly to subsequent challenge with 25-hydroxycholesterol. Finally, we measured the ability of the two reductase inhibitors to reduce ApoB secretion by HepG2 cells. Simva and Atorva at 0.5 microM inhibited ApoB secretion nearly identically, 38% and 42% respectively. We conclude that these two drugs induce similar adaptive responses in cells and that their actions are qualitatively and mechanistically identical. Human studies have shown that plasma is cleared of Atorva much more slowly than it is of Simva. The large pharmacokinetic difference in man, rather than some difference in mechanism, is the most likely explanation for the finding that the equipotent dose ratio for cholesterol lowering in humans of Simva to Atorva is about 2/1.
Assuntos
Colesterol/biossíntese , Ácidos Heptanoicos/farmacologia , Hidroximetilglutaril-CoA Redutases/biossíntese , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fígado/enzimologia , Pirróis/farmacologia , Sinvastatina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Anticolesterolemiantes/farmacologia , Atorvastatina , Carcinoma Hepatocelular , Indução Enzimática , Humanos , Cinética , Fígado/efeitos dos fármacos , Neoplasias Hepáticas , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.
Assuntos
Alendronato/farmacologia , Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Animais , Isomerases de Ligação Dupla Carbono-Carbono/antagonistas & inibidores , Difosfonatos/farmacologia , Farnesiltranstransferase , Geraniltranstransferase , Hemiterpenos , Humanos , Técnicas In Vitro , Cinética , Lipídeos/biossíntese , Fígado/enzimologia , Masculino , Ácido Mevalônico/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Pamidronato , Prenilação de Proteína/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/antagonistas & inibidoresRESUMO
Tissue selectivity of lovastatin, simvastatin and pravastatin was determined in male rats. Peak levels of active drug were found in all tissues examined between 0.5 and 2 hours after oral administration. The area under the curve describing 24 hour exposure of the tissues to drug indicated that the drugs were preferentially concentrated in the liver. However, the concentration of pravastatin was approximately 50% that of either lovastatin or simvastatin in the liver and 3-6 times higher in peripheral tissues. These studies demonstrate that the hydrophobic prodrugs, lovastatin and simvastatin show greater selectivity than the hydrophilic agent pravastatin towards the liver which is the target organ for inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
Assuntos
Ácidos Heptanoicos/farmacocinética , Lovastatina/análogos & derivados , Lovastatina/farmacocinética , Naftalenos/farmacocinética , Glândulas Suprarrenais/metabolismo , Animais , Mucosa Gástrica/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases , Rim/metabolismo , Fígado/metabolismo , Masculino , Pravastatina , Ratos , Sinvastatina , Baço/metabolismo , Testículo/metabolismo , Distribuição TecidualRESUMO
A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.
Assuntos
Dimetilaliltranstransferase/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Transferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA/genética , Dimetilaliltranstransferase/biossíntese , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RatosRESUMO
Farnesyl diphosphate, the substrate for squalene synthase, accumulates in the presence of zaragozic acid A, a squalene synthase inhibitor. A possible metabolic fate for farnesyl diphosphate is its conversion to farnesol, then to farnesoic acid, and finally to farnesol-derived dicarboxylic acids (FDDCAs) which would then be excreted in the urine. Seven dicarboxylic acids were isolated by high performance liquid chromatography (HPLC) from urine of either rats or dogs treated with zaragozic acid A or rats fed farnesol. Their structures were determined by nuclear magnetic resonance analysis. Two 12-carbon, four 10-carbon, and one 7-carbon FDDCA were identified. The profile of urinary dicarboxylic acids from rats fed farnesol was virtually identical to that produced by treating with zaragozic acid A, establishing that these dicarboxylic acids are farnesol-derived. By feeding [1-14C]farnesol and comparing the mass of the dicarboxylic acids produced with the ultraviolet absorption of the HPLC peaks, a method to quantitate the ultraviolet-absorbing FDDCAs was devised. When rats were treated with zaragozic acid A, large amounts of FDDCAs were excreted in the urine. The high level of FDDCAs that were found suggests that their synthesis is the major metabolic fate for carbon diverted from cholesterol synthesis by a squalene synthase inhibitor. A metabolic pathway is proposed to explain the production of each of these FDDCAs.
Assuntos
Anticolesterolemiantes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ácidos Dicarboxílicos/urina , Farneseno Álcool/farmacologia , Ácidos Tricarboxílicos/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Cães , Farneseno Álcool/urina , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Espectroscopia de Ressonância Magnética , Modelos Químicos , Ratos , Espectrofotometria UltravioletaRESUMO
Three closely related fungal metabolites, zaragozic acids A, B, and C, that are potent inhibitors of squalene synthase have been isolated and characterized. Zaragozic acids A, B, and C were produced from an unidentified sterile fungal culture, Sporormiella intermedia, and Leptodontium elatius, respectively. The structures of the zaragozic acids and their trimethyl esters were determined by a combination of physical and chemical techniques. The zaragozic acids are characterized by a novel 2,8-dioxobicyclo[3.2.1]octane-4,6,7- trihydroxyl-3,4,5-tricarboxylic acid core and differ from each other in the structures of the 6-acyl and 1-alkyl side chains. They were found to be potent competitive inhibitors of rat liver squalene synthase with apparent Ki values of 78 pM, 29 pM, and 45 pM, respectively. They inhibited cholesterol synthesis in Hep G2 cells, and zaragozic acid A was an inhibitor of acute hepatic cholesterol synthesis in the mouse (50% inhibitory dose of 200 micrograms/kg of body weight). Inhibition of squalene synthase in cells and in vivo was accompanied by an accumulation of label from [3H]mevalonate into farnesyl diphosphate, farnesol, and organic acids. These data indicate that the zaragozic acids are a previously unreported class of therapeutic agents with potential for the treatment of hypercholesterolemia.