Cholesterotropic and cuprotropic chemicals.

A dozen or so chemicals modify both cholesterol and copper metabolism. Ascorbic acid and cadmium, etc., inhibit copper metabolism and raise cholesterol. Calcium and clofibrate, etc., enhance copper and lower cholesterol. Perhaps the doses of dietary cholesterol and fructose in this experiment were too severe to permit fenofibrate to lower cholesterol in a manner similar to clofibrate. © 2020 Society of Chemical Industry.

Clofibrate relaxes the longitudinal smooth muscle of the mouse distal colon through calcium-mediated desensitisation of contractile machinery.

Peroxisome proliferator-activated receptor α (PPAR-α) is a ligand-activated transcription factor that exerts strong effects on metabolic pathways. Our aim was to elucidate the effect of clofibrate, a PPAR-α agonist, on the longitudinal muscle of the mouse distal colon. We initially found that clofibrate induced a relaxation response in this muscle. Notably, the PPAR-α antagonists GW9662 and T0070907 did not attenuate this clofibrate-induced relaxation. The structurally related PPAR-α agonists fenofibrate and bezafibrate induced relaxation in the distal colon as effectively as clofibrate. In contrast, wy-14643, which activates PPAR-α more selectively than clofibrate, had no effect. Furthermore, clofibrate-induced relaxation was not affected by N-nitro-L-arginine, an NO synthase inhibitor, 1H-[1,2,4]-oxadiazolo-[4,3- a]quinoxaline-1-one, a soluble guanylate cyclase inhibitor, or H89, a protein kinase A inhibitor. Tetrodotoxin, an Na⁺ channel blocker, and glibenclamide, apamin, charybdotoxin and XE991, various K⁺ channel blockers, had no effect on clofibrate-induced relaxation. Importantly, clofibrate induced a relaxation response that was not accompanied by any alteration in the cytoplasmic Ca²âº concentration in the longitudinal muscle of the mouse distal colon. Moreover, calyculin A, a myosin light-chain phosphatase (MLCP) inhibitor, attenuated clofibrate-induced relaxation. Our findings indicate that clofibrate relaxes the longitudinal smooth muscle of the mouse distal colon by regulating MLCP activity.

Is the metabolic syndrome an intracellular Cushing state? Effects of multiple humoral factors on the transcriptional activity of the hepatic glucocorticoid-activating enzyme (11beta-hydroxysteroid dehydrogenase type 1) gene.

Although glucocorticoid, as "gluco-" literally implies, plays an important role in maintaining the blood glucose level, excess of glucocorticoid production/action is known to cause impaired glucose tolerance and diabetes. Since 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which converts inactive cortisone to active cortisol, is primarily expressed in the liver, an enhanced expression of the enzyme may increase the intracellular glucocorticoid level and thus increase the hepatic glucose production. In this study, we examined the effects of multiple humoral factors related to the metabolic syndrome on the transcriptional activity of 11beta-HSD1 gene in hepatocytes in vitro. We found that, among the factors examined, adipocyte-derived cytokines (adipokines), like TNFalpha and IL-1beta, potently stimulated the transcriptional activity of 11beta-HSD1 gene in human HuH7 cells. In contrast, only minimal effects of other humoral factors were observed when they were used alone. Interestingly, however, when applied in combination, they synergistically enhanced the transcriptional activity of 11beta-HSD1 gene. They also potentiated the effects of cytokines. Glucocorticoid receptor (GR)-dependent transcription was indeed increased even with an inactive glucocorticoid cortisone following TNFalpha pretreatment, indicating the enhanced intracellular conversion. Finally, PPARgamma/PPARalpha agonists, clinically used as anti-diabetic drugs, significantly inhibited the transcriptional activity of 11beta-HSD1. Altogether, our data strongly suggest that combination of the humoral factors related to the metabolic syndrome, including the adipokines, synergistically enhances the hepatic expression of 11beta-HSD1 gene and causes the intracellular Cushing state in the liver by increasing the intracellular glucocorticoid level. We assume that the observed synergistic effects of these factors on 11beta-HSD1 may, at least partly, explain the reason whereby accumulation of the multiple risk factors facilitates the derangement of glucose and lipid metabolism in the metabolic syndrome.

Pharmacokinetics of drugs in patients with the nephrotic syndrome.

Since the binding of drugs to plasma proteins can significantly after the intensity of pharmacological and toxicological effects of drugs, we studied the pharmacokinetics of three drugs in patients with hypoalbuminemia secondary to the nephrotic syndrome, but with relatively normal renal function. No significant differences were seen in the pharmacokinetic parameters observed for antipyrine, a drug which is less than 10% bound to plasms proteins. The percentage of unbound diphenylhydantoin, a highly plasms protein-bound drug, was found in patients with the nephrotic syndrome to be twice that of healthy individuals (19,2 vs. 10.1%, P smaller than 0.001). However, there was also a lower steady-state plasma concentration of diphenylhydantoin (2.9 plus or minus 0.6 vs. 6.8 plus or minus 0.6 mug/ml, P smaller than 0.001) secondary to an increase in the plasms clearance (0.048 plus or minus 0.019 vs. 0.022 plus or minus 0.006 liter/kg.h, P smaller than 0.001) in the nephrotic patients. The net effect is no difference in the absolute concentration of unbound diphenylhydantoin in healthy individuals (0.69 plus or minus 0.05 mug/ml) and patients with the nephrotic syndrome (0.59 plus or minus 0.06 mug/ml). Qualitatively, similar differences were observed with clofibrate. The dose of these drugs need not be routinely reduced in patients with the nephrotic syndrome as long as they have reasonably normal renal function (creatinine clearance greater than 50 ml/min). With all highly bound acidic drugs, knowledge of the concentration of unbound drug is essential to the proper interpretation of total blood levels and subsequent treatment of the patient.

Peroxisome proliferator-activated receptor alpha-retinoid X receptor agonists induce beta-cell protection against palmitate toxicity.

Fatty acids can stimulate the secretory activity of insulin-producing beta-cells. At elevated concentrations, they can also be toxic to isolated beta-cells. This toxicity varies inversely with the cellular ability to accumulate neutral lipids in the cytoplasm. To further examine whether cytoprotection can be achieved by decreasing cytoplasmic levels of free acyl moieties, we investigated whether palmitate toxicity is also lowered by stimulating its beta-oxidation. Lower rates of palmitate-induced beta-cell death were measured in the presence of L-carnitine as well as after addition of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, conditions leading to increased palmitate oxidation. In contrast, inhibition of mitochondrial beta-oxidation by etomoxir increased palmitate toxicity. A combination of PPARalpha and retinoid X receptor (RXR) agonists acted synergistically and led to complete protection; this was associated with enhanced expression levels of genes involved in mitochondrial and peroxisomal beta-oxidation, lipid metabolism, and peroxisome proliferation. PPARalpha-RXR protection was abolished by the carnitine palmitoyl transferase 1 inhibitor etomoxir. These observations indicate that PPARalpha and RXR regulate beta-cell susceptibility to long-chain fatty acid toxicity by increasing the rates of beta-oxidation and by involving peroxisomes in fatty acid metabolism.

The biochemistry of hypo- and hyperlipidemic fatty acid derivatives: metabolism and metabolic effects.

A selection of amphipatic hyper- and hypolipidemic fatty acid derivatives (fibrates, thia- and branched chain fatty acids) are reviewed. They are probably all ligands for the peroxisome proliferation activation receptor (PPARalpha) which has a low selectivity for its ligands. These compounds give hyper- or hypolipidemic responses depending on their ability to inhibit or stimulate mitochondrial fatty acid oxidation in the liver. The hypolipidemic response is explained by the following metabolic effects: Lipoprotein lipase is induced in liver where it is normally not expressed. Apolipoprotein CIII is downregulated. These two effects in liver lead to a facilitated (re)uptake of chylomicrons and VLDL, thus creating a direct transport of fatty acids from the gut to the liver. Fatty acid metabolizing enzymes in the liver (CPT-I and II, peroxisomal and mitochondrial beta-oxidation enzymes, enzymes of ketogenesis, and omega-oxidation enzymes) are induced and create an increased capacity for fatty acid oxidation. The increased oxidation of fatty acids "drains" fatty acids from the body, reduces VLDL formation, and ultimately explains the antiadiposity and improved insulin sensitivity observed after administration of peroxisome proliferators.

A metabonomics study of the hepatotoxicants galactosamine, methylene dianiline and clofibrate in rats.

The efficacy of high-resolution (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy-based metabonomics was studied in a model of rat liver toxicity. Hepatotoxicities were induced in male rats using methylene dianiline, clofibrate and galactosamine. Twenty-four-hr urine from days 1 to 5 after treatment were subjected to (1)H-NMR evaluation of the biochemical effects. Blood were also taken at Days 2, 3 and 5 to examine biochemical changes associated with hepatotoxicities, and histopathological changes were evaluated at termination. Increases in liver enzymes were observed in animals treated with methylene dianiline or galactosamine, and histopathological analysis revealed changes associated with hepatobiliary damage and hepatocellular necrosis in methylene dianiline- and galactosamine-treated animals, respectively. Principal component analysis and statistical Spotfire analyses were used to visualize similarities and differences in urine biochemical profiles produced by (1)H-NMR spectra. The biochemical effects of methylene dianiline and galactosamine were characterized by elevated levels of glucose, fructose, beta-hydroxybutyrate, alanine, acetoacetate, lactate and creatine and decreased levels of hippurate, 2-oxoglutarate, citrate, succinate, trimethylamine-N-oxide, taurine and N-acetylglutamate in rat urine. Clofibrate treatment elevated the levels of N-methylnicotinamide and 3,4-dihydroxymandelate and decreased the levels of 2-oxoglutarate and N-acetylaspartate. This work shows that combinations of (1)H-NMR and pattern recognition are powerful tools in the evaluation of the biochemical effects of xenobiotics in liver.

Binding of spin-labeled clofibrate to lipoproteins.

The binding of spin-labeled clofibrate to native and partially delipidated lipoproteins is a rapid, linear and non-saturable process observed up to the critical micellar concentration of the drug. Low-density lipoproteins (LDL) display a lower affinity for the drug than very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) relative to their respective specific volume. Unlike various lipophilic drugs, uptake of spin-labeled clofibrate does not correlate with lipoprotein lipid volume. Spin-labeled clofibrate binding to LDL is enhanced when the temperature increases above 25 degrees C. The binding to HDL and VLDL is less temperature-sensitive. The simulation of the ESR spectra has shown that two types of motion should be superimposed for the spin-labeled clofibrate in HDL, in LDL or in partially delipidated LDL. From 40 down to 25 degrees C for HDL and LDL, a fast anisotropic motion is observed. From 25 degrees C down to 5 degrees C, a two-component motion takes place, including a slow isotropic motion of the probe tumbling in a highly hydrophobic environment. Interactions of spin-labeled clofibrate with the apolipoproteins in HDL and LDL are assumed from the emergence of this strongly immobilized component observed when the temperature decreases. In contrast, for spin-labeled clofibrate inserted in the apolar core of VLDL, ESR shows only one component in the whole temperature range (5-40 degrees C). The location of the spin-labeled drug inside the various lipoprotein particles is discussed as a function of temperature.

[Pharmacology of fibrates]. / Farmacologia do fibratos.

Fibrates are drugs commonly used in hypertriglyceridemias. After their absorption, fibrates are metabolized by the liver, using P450 isoenzymes not shared by statins. However, for some fibrates, such as genfibrozil, an interaction with statins can occur during their liver glucuronidation, or by displacement of free fraction of statins linked to proteins in plasma. Plasma half-life is variable among fibrates (2-80 h). Recently it has been shown that fibrates produce plasma lipid changes mainly by stimulating peroxisome-proliferation activation receptors alpha. Through this action, there is an increase in the transcription of some genes related to lipid metabolism, such as LLP, APOAI, APOAII, ABCA-1, as well as decrease in the expression of APOCIII, and many other actions.

[Mechanisms of hepatotoxicity]. / Mecanismos de hepatotoxicidade.

Liver disease following the use of hypolipidemic drugs has been reported as a cellular damage (increases in AST or ALT enzymes) without cholestatic alterations (bilirubin and or alkaline phosphatase increases). Six mechanisms were proposed for hepatotoxicity: 1. High energy reactions on P450 cytochrome impairing calcium homeostasis with rupture of intracellular fibrils and hepatocyte lysis. 2. Impairment of transporter proteins related to the bile acids flux (mechanism proposed for fibrate liver toxicity). 3. Immune reactions due to the formation of metabolites linked to enzymes following liver metabolism of hypolipidemic drugs. 4. Hepatotoxicity by T cells with additional inflammation mediated by neutrophils. 5. Apoptosis mediated by TNF and Fas (immune mediated). 6. Oxidative stress due to damage of intracellular organelles. In addition, advanced age, alcohol in excess, high doses of hypolipidemic drugs, interaction with other drugs, and previous active liver disease might increase liver toxicity.

[Hypolipidemic therapy in special situations: hypothyroidism and liver disease]. / Terapia hipolipemiante em situações especiais--hipotireoidismo e hepatopatias.

Hypothyroidism is common in the elderly, especially among women. It should be suspected in the presence of classic signals and symptoms, and can be detected by an elevation of serum thyroid stimulating hormone (TSH). Lipid abnormalities in the presence of subclinical hypothyroidism are of minor importance. However, the importance of specific treatment (hormone replacement) increases with the magnitude of thyroid disturbance. Some hypolipidemic agents can aggravate prior liver disease, however, recent studies have shown that statins might be useful in the presence of steatohepatitis. Some associations of hypolipidemic drugs can increase liver enzymes, and careful monitoring is recommended.

Clofibrate kinetics after single and multiple doses.

The kinetics of chlorphenoxyisobutyric acid (CPIB) were studied in 5 healthy subjects after single 500-mg, 1,000-mg, and 2,000-mg doses of clofibrate, and in steady-state after 8 days' treatment with 1,000 mg twice daily. Maximum plasma concentrations of CPIB were observed 4 to 6 hr after dosing. A mean plasma half-life of 16.7 hr was recorded which was independent of dose and duration of treatment. Total plasma clearance (-Cl) calculated from area under the curve with the use of the total plasma concentration was 5.6 ml/min for the 500-and the 1,000-mg doses but increased to 6.8 ml/min for the 2,000-mg dose and was even higher (8.1 ml/min) in steady-state. This change in -Cl is a consequence of progressive reduction in the plasma protein binding of clofibrate at plasma concentrations above 50 microgram/ml, since -Cl rises in association with reduced protein binding at the high plasma concentrations measured after the 2,000-mg single dose and in steady-state. -Cl and apparent volume of distribution were identical for all doses tested when calculations were based on the nonprotein-bound CPIB concentrations only. Due to the inconsistant protein binding of CPIB, total steady-state concentrations could not be predicted from the single dose kinetic data.

Probenecid-clofibrate interaction.

Clofibric acid disposition was studied in four healthy men after 1 wk of clofibrate ingestion (500 mg orally every 12 hr) with and without probenecid (500 mg orally every 6 hr). Mean (+/- SD) free clofibric acid plasma concentration in the four subjects over a dosage interval at steady state was 2.5 +/- 0.03 mg/1 before and 9.05 +/- 1.09 mg/1 after the probenecid. Probenecid reached an average plasma concentration of 71.3 mg/1. No clofibric acid glucuronide was detected in plasma during either treatment. The fractions of the dose recovered in urine as clofibric acid, clofibric acid glucuronide, and clofibric acid liberated after acid hydrolysis were not altered by probenecid. These data suggest that probenecid causes a reduction in renal and metabolic clearance of clofibric acid, probably as a result of inhibition of the conjugation of clofibric acid with glucuronide.

Protective effects of clofibrate on isoproterenol-induced myocardial infarction in arteriosclerotic and non-arteriosclerotic rats.

Male and female, arteriosclerotic and non-arteriosclerotic rats were treated with the anti-lipemic agent, clofibrate, for 8 days and then subjected to an acute myocardial infarction by injecting them with two large doses of isoproterenol spaced 24 hours apart. The animals were killed at sequential time intervals during the acute necrosis and early repair phases of myocardial infarction. Pre-treatment with clofibrate caused a definite improvement in survival, less shock and prostration, and ECG evidence of little or no ischemia. Increased SGOT levels, hepatic lipid and necrosis were indicative of advanced liver damage. Although clofibrate-treated animals showed little change in serum lipids during the acute cardiac necrosis phase, they were hyperglycemic and showed the greatest increase in BUN levels. Clofibrate-treated animals had higher serum corticosterone levels than those given isoproterenol alone. Despite superior survival rates, both the arteriosclerotic and non-arteriosclerotic, clofibrate-treated animals exhibited equally severe histopathologic evidence of myocardial damage. It is suggested that the protective effect of prophylactic treatment with clofibrate against isoproterenol-induced myocardial infarction in rats may be due to its ability to change corticosterone levels in the circulation.

Active drug metabolites and renal failure.

Drugs that are administered to man may be biotransformed to yield metabolites that are pharmacologically active. These metabolites may accumulate in patients with end-stage renal disease if renal excretion is a major elimination pathway for the metabolite. This is true even if the active metabolite is a minor metabolite of the parent drug as long as the minor metabolite is not further biotransformed but is mainly excreted in the urine. Minor metabolite accumulation may also occur if it is further biotransformed by a pathway that is inhibited in uremia. Some clinical consequences of accumulation of the active drug metabolites of procainamide, meperidine, clofibrate, allopurinol, sulfadiazine and nitrofurantoin in patients with renal failure are discussed. The high incidence of adverse drug reactions seen in renal failure may be explained, in part, by the accumulation of active drug metabolites. Examples of active drug metabolites that do not accumulate in patients with renal failure because of further biotransformations are also included.

Pharmacologically active drug metabolites: therapeutic and toxic activities, plasma and urine data in man, accumulation in renal failure.

Drugs that are administered to man may be biotransformed to yield metabolites that are pharmacologically active. The therapeutic and toxic activities of drug metabolites and the species in which this activity was demonstrated are compiled for the metabolites of 58 drugs. The metabolite to parent drug ratio in the plasma of non-uraemic man and the percentage urinary excretion of the metabolite in non-uraemic man are also tabulated. Those active metabolites with significant pharmacological activity and high plasma levels, both relative to that of the parent drug, will probably contribute substantially to the pharmacological effect ascribed to the parent drug. Active metabolites may accumulate in patients with end stage renal disease if renal excretion is a major elimination pathway for the metabolite. This is true even if the active metabolite is a minor metabolite of the parent drug, as long as the minor metabolite is not further biotransformed and is mainly excreted in the urine. Minor metabolite accumulation may also occur if it is further biotransformed by a pathway inhibited in uraemia. Some clinical examples of the accumulation of active drug metabolites in patients with renal failure are: (a) The abolition of premature ventricular contractions and prevention of paroxysmal atrial tachycardia in some cardiac patients with poor renal function treated with procainamide are associated with high levels of N-acetylprocainamide. (b) The severe irritability and twitching seen in a uraemic patient treated with pethidine (meperidine) are associated with high levels of norpethidine. (c) The severe muscle weakness and tenderness seen in patients with renal failure receiving clofibrate are associated with excessive accumulation of the free acid metabolite of clofibrate. (d) Patients with severe renal insufficiency taking allopurinol appear to experience a higher incidence of side reactions, possibly due to the accumulation of oxipurinol. (e) Accumulation of free and acetylated sulphonamides in patients with renal failure is associated with an increase in toxic side-effects (severe nausea and vomiting, evanescent macular rash). (f) Peripheral neuritis seen after nitrofurantoin therapy in patients with impaired renal function is thought to be due to accumulation of a toxic metabolite. The high incidence of adverse drug reactions seen in patients with renal failure may for some drugs be explained in part, as the above examples illustrate, by the accumulation of active drug metabolites. Monitoring plasma levels of drugs can be an important guide to therapy. However, if a drug has an active metabolite, determination of parent drug alone may cause misleading interpretations of blood level measurements. The plasma level of the active metabolite should also be determined and its time-action characteristics taken into account in any clinical decisions based on drug level monitoring.

Clinical significance of esterases in man.

Esterases, hydrolases which split ester bonds, hydrolyse a number of compounds used as drugs in humans. The enzymes involved are classified broadly as cholinesterases (including acetylcholinesterase), carboxylesterases, and arylesterases, but apart from acetylcholinesterase, their biological function is unknown. The acetylcholinesterase present in nerve endings involved in neurotransmission is inhibited by anticholinesterase drugs, e.g. neostigmine, and by organophosphorous compounds (mainly insecticides). Cholinesterases are primarily involved in drug hydrolysis in the plasma, arylesterases in the plasma and red blood cells, and carboxylesterases in the liver, gut and other tissues. The esterases exhibit specificities for certain substrates and inhibitors but a drug is often hydrolysed by more than one esterase at different sites. Aspirin (acetylsalicylic acid), for example, is hydrolysed to salicylate by carboxylesterases in the liver during the first-pass. Only 60% of an oral dose reaches the systemic circulation where it is hydrolysed by plasma cholinesterases and albumin and red blood cell arylesterases. Thus, the concentration of aspirin relative to salicylate in the circulation may be affected by individual variation in esterase levels and the relative roles of the different esterases, and this may influence the overall pharmacological effect. Other drugs have been less extensively investigated than aspirin and these include heroin (diacetylmorphine), suxamethonium (succinylcholine), clofibrate, carbimazole, procaine and other local anaesthetics. Ester prodrugs are widely used to improve absorption of drugs and in depot preparations. The active drug is released by hydrolysis by tissue carboxylesterases. Individual differences in esterase activity may be genetically determined, as is the case with atypical cholinesterases and the polymorphic distribution of serum paraoxonase and red blood cell esterase D. Disease states may also alter esterase activity.

Hydrolysis of ester- and amide-type drugs by the purified isoenzymes of nonspecific carboxylesterase from rat liver.

Five purified carboxylesterases from rat liver microsomes show a differing capacity for the hydrolysis of ester- and amide-type drugs. The two closely related enzymes that are responsible for the microsomal hydrolysis of palmitoyl-CoA and long chain monoacylglycerides exhibit the highest propanidid-and aspirin-cleaving rates. The predominant nonspecific esterase of microsomes is responsible for the hydrolysis of procaine, clofibrate, isoarecaidine esters, butanilicaine, octanoylamide, and possibly butyryl thiocholine. Finally, the palmitoyl carnitine-cleaving esterase splits phenacetin and acetanilide. The purified nonspecific esterase with the lowest isoelectric point is not involved in the metabolism of the drugs mentioned.

The effect of colestipol hydrochloride on the bioavailability and pharmacokinetics of clofibrate.

Since it has been reported by several authors that colestipol HCl and clofibrate have an additive effect in lowering serum cholesterol levels, it was felt advisable to evaluate the blood levels of clofibrate when given simultaneously with colestipol HCl to see whether there was any evidence for drug interaction between the two products that might dictate a need for separation of their administration time. After concomitant single-dose administration, the serum p-chlorophenoxyisobutyric acid levels, bioavailability parameters, and pharmacokinetic parameters investigated provided no evidence for an interaction and suggested that colestipol and clofibrate can be administered concomitantly or at separated in tervals according to whichever dosage regimen is deemed advisable by the physician.

Lack of covalent binding to rat liver DNA of the hypolipidemic drugs clofibrate and fenofibrate.

14C-labelled clofibric acid and fenofibric acid were administered p.o. to 200 g male and female rats. After 10 h, liver nuclear DNA and protein were isolated and the radioactivity was determined. Binding to protein was clearly measurable whereas no binding to DNA could be detected from any drug. A comparison of the limit of detection of such DNA binding with well-known chemical carcinogens revealed that the known hepatocarcinogenicity of clofibrate cannot be based upon an initiating, DNA damaging, mode of action but must be due to other, nongenotoxic, mechanisms such as peroxisome proliferation, hepatomegaly, or cytotoxicity due to protein binding. The risk assessment in man and the interpretation of the carcinogenicity data for rodents are discussed.