Determination of minalrestat (an aldose reductase inhibitor) in rat, dog and human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method was developed for the quantification of minalrestat (a potent aldose reductase inhibitor) in rat, dog and human plasma. Minalrestat and internal standard (I.S.) were extracted from plasma by either solid-phase extraction (SPE) or liquid-liquid extraction (human plasma). Plasma extracts were chromatographed on a Hypersil ODS column with 3-microm packing with a mobile phase of acetonitrile-0.05 M potassium phosphate buffer, pH 3.0 (45:55, v/v) at 0.2 ml/min. The signal in the eluent was enhanced by UV-irradiation when passing through a photochemical reaction unit with a 10-m reaction coil, prior to detection by UV absorbance at 255 nm. The intra-day coefficients of variation was less than 9% in rat, dog and human plasma and the intra-day accuracy (%MRE) was within +/-5% in all matrices tested. The inter-day coefficients of variation were less than 12% in rat and human plasma and the accuracy (%MRE) was within +/-15%. Minalrestat was stable for at least 60 days in rat and human plasma and at least 30 days in dog plasma samples stored at -20 degrees C. In human plasma samples, the analyte was stable for up to 5 cycles of freezing and thawing. This method has been applied successfully for the evaluation of the pharmacokinetics of minalrestat in rats, dogs and humans.

Lack of protection of PBN in isolated heart during ischemia and reperfusion: implications for radical scavenging mechanism.

We evaluated the ability of alpha-phenyl-tert-butyl nitrone (PBN) to trap free radicals and to protect the rat myocardium during ischemia and reperfusion. Isolated bicarbonate buffer-perfused hearts (n = 8) were subjected to 20 min global ischemia (37 degrees C) followed by reperfusion with 0.4 to 4.0 mM PBN. Coronary effluent containing the PBN adduct was extracted in toluene. Electron spin resonance analysis of the toluene extract revealed a PBN-hydroxyl adduct. To verify this assignment, a Fenton system was used to generate an authentic PBN-hydroxyl adduct (n = 8), which yielded the same ESR spectra as the reperfusion-derived adduct. The structure of the adduct formed in the Fenton system was confirmed by gas chromatography-mass spectrometry. The ESR parameters of the PBN-hydroxyl adduct were exquisitely sensitive to solvent polarity during extraction of the adduct. Extraction of an authentic PBN-hydroxyl adduct into chloroform, chloroform:methanol, and toluene closely matched the ESR parameters obtained during reperfusion of ischemic myocardium in other animal models. To determine whether PBN could confer any protective effect during ischemia or reperfusion, hearts (n = 8/group) were subjected to 35 min global ischemia at 37 degrees C with the St. Thomas' II cardioplegic solution followed by 30 min reperfusion. Percent recovery (mean +/- SEM) of developed pressure, rate pressure product, and leakage of lactate dehydrogenase during reperfusion in control hearts were 58 +/- 3%, 48 +/- 4% and 3.2 +/- 0.5 IU/15 min/g wet wt. PBN at a concentration of 0.4 mM or 4.0 mM when present either during ischemia alone or reperfusion alone did not exert any effect upon recovery of developed pressure, rate pressure product or post-ischemic enzyme leakage. We conclude that PBN fails to improve contractile recovery and reduce enzyme leakage during reperfusion of myocardium subjected to global ischemia.

Cholinomimetic compound distinct from caffeine contained in coffee. II: Muscarinic actions.

An extract with cholinergic activities was isolated from instant regular and decaffeinated coffees and purified. Intravenous injection of this cholinomimetic extract of coffee produced an abrupt depression in blood pressure and heart rate, changes that were distinct from those of known components of coffee, including caffeine, trigonelline, catechin, and chlorogenic acid. Pretreatment of the animals with naloxone, propranolol, isobutylmethylxanthine, hexamethonium bromide, and hemicholinium-3 chloride or bilateral vagotomy did not affect the cardiodepressive effects of the extract, whereas atropine completely abolished them. Direct injection of the cholinomimetic extract of coffee (20-100 micrograms) into the periaqueductal gray area of the midbrain did not produce any cardiovascular effect. However, the extract of coffee did cause relaxation of isolated rat and rabbit aortic ring preparations that were contracted under norepinephrine. The cholinomimetic extract did not inhibit purified acetylcholinesterase. This pharmacologic profile indicates that the cholinomimetic extract of coffee acts as a direct muscarinic agonist.

Coffee contains cholinomimetic compound distinct from caffeine. I: Purification and chromatographic analysis.

Both regular and decaffeinated coffees were found to have cholinomimetic actions when tested in urethane-anesthetized rats. These actions were distinct from those of caffeine and reversible by atropine. The bioactive fraction was purified from alcoholic extracts of instant decaffeinated coffee by liquid column chromatography and preparative TLC. The purified compound showed similar pharmacological actions as the starting material. Chromatographic behavior was further characterized by analytical TLC and HPLC. Chromatographic analyses of extracts of green coffee beans and roasted ground coffees showed that the cardioactive compound was only present in roasted coffees. Similar analyses of other commonly consumed beverages, including teas and cocoa, showed that this compound was not present in beverages besides coffee.

Antioxidative properties of harmane and beta-carboline alkaloids.

beta-Carboline alkaloids are derived as a result of condensation between indoleamine (e.g. tryptamine) and short-chain carboxylic acid (e.g. pyruvic acid) or aldehyde (e.g. acetaldehyde), a reaction that occurs readily at room temperature. These compounds have been found endogenously in human and animal tissues and may be formed as a byproduct of secondary metabolism: their endogenous functions however, are not well understood. Indoles and tryptophan derivatives exhibit antioxidative actions by scavenging free radicals and forming resonance stabilized indolyl radicals. Harmane and related compounds exhibited concentration-dependent inhibition of lipid peroxidation (measured as thiobarbiturate reactive products) in a hepatic microsomal preparation incubated with either enzymatic dependent (Fe3+ ADP/NADPH) or non-enzymatic dependent (Fe3+ ADP/dihydroxyfumarate) oxygen radical producing systems. Alkaloids with hydroxyl substitution and a partially desaturated pyridyl ring were found to have the highest antioxidative potencies. Substitution of a hydroxyl group by a methoxyl group at the 6-position resulted in a decrease of greater than 10-fold in the antioxidative activities. Harmane showed high efficacy in an enzymatic system but low efficacy in a non-enzymatic system. The antioxidative effects of harmane in the former system may be attributed to its ability to inhibit oxidative enzymes in the microsomal system. These results suggest that beta-carbolines may also serve as endogenous antioxidants.

Chlorinated aliphatic hydrocarbons promote lipid peroxidation in vascular cells.

Chlorinated aliphatic hydrocarbons such as trichloroethylene and trichloroethane cause acute and chronic cardiotoxicities via mechanisms that are still obscure. Some of these toxic effects may result from free-radical-induced membrane damage to the vascular tissues. The pro-oxidant effects of carbon tetrachloride, trichloroethylene, 1,1,1-trichloroethane, 1,2-dichloroethane, and trans-1,2-dichloroethylene were assessed in cultured arterial endothelial and aortic smooth muscle cells. Exposure of the cells to the above chemicals alone did not increase the formation of thiobarbiturate reactive products above background levels. However, in the presence of low levels of iron (3.1-25 microM Fe3+ chelated by ADP), all five agents promoted lipid peroxidation up to 200% of control. The rank order of potency is carbon tetrachloride greater than or equal to trichloroethylene greater than 1,1,1-trichloroethane greater than trans-1,2-dichloroethylene greater than 1,2-dichloroethane. The synergistic interaction between iron and chlorinated hydrocarbons in promoting lipid peroxidation may contribute to the cardiotoxicities of these agents.

Oxygen radical-mediated injury of myocytes-protection by propranolol.

UIe effects of propranolol and atenolol on free radical mediated injury in myocytes were examined. Freshly isolated adult canine myocytes were incubated with a superoxide generating (from dihydroxyfumarate) and Fe-catalyzed free radical system. Exposure of the myocytes to free radicals for 20 min resulted in more than a 5-fold increase in thiobarbituric acid reactant (peroxide) formation and elevated levels of lactate dehydrogenase (LDH) activity released into the media compared to controls. Ultrastructurally, severe sarcolemmal damage, mitochondrial and myofibril derangements were evident. At 40 min, cellular viability (trypan blue exclusion) in the samples exposed to free radicals decreased to about one-third of controls; concomitantly, major losses in total cellular phospholipids occurred. When the cells were pretreated with 200 microM propranolol before the addition of free radicals, both peroxide formation and increased LDH release were inhibited; in agreement, complete ultrastructural preservation was observed. In addition, the subsequent losses in cellular viability and phospholipids were prevented. For comparison, the more water soluble beta-blocker, atenolol at 200 microM was shown ineffective in providing significant protection against the induced injury. The results suggest that propranolol may provide antiperoxidative protection to myocytes when elevated levels of free radicals are present.

Inhibition of the shake response in rats by adenosine and 2-chloroadenosine.

Shaking movements, similar to those made by a dog when wet, were elicited in rats by immersion in ice-water, injections of icilin, a chemical that produces sensations of cold, and naloxone-precipitated morphine withdrawal. Adenosine and 2-chloroadenosine produced dose-dependent inhibition of shaking to ice-water and icilin. The 2-chloroadenosine effect was mediated centrally because the ICV dose required to produce inhibition was not effective when given IP. Caffeine antagonized the inhibitory effects of adenosine and 2-chloroadenosine. 2-Chloroadenosine suppressed morphine-abstinence shaking as well as the body weight loss that normally accompanies withdrawal.

Cardiovascular effects of intraventricular injection of FMRFamide, Met-enkephalin and their common analogues in the rat.

The molluscan neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide), the mammalian opioid peptide met-enkephalin, and their common analogues, met-enkephalin-Arg6-Phe7 (YGGFMRF) and Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide (YGGFMRFamide), were injected into the lateral ventricle of the rat; the cardiovascular effects were studied. FMRFamide caused a rapid, transient elevation in blood pressure accompanied by a great increase in pulse pressure. These effects were followed by secondary increases in blood and pulse pressures. Met-enkephalin produced an initial reduction in blood pressure which was followed by a gradual increase at the higher of two test doses (300 nmole). Injection of YGGFMRF resulted in a gradual increase in blood pressure. This response resembled that to met-enkephalin. The initial response to YGGFMRFamide was similar to that to FMRFamide: increases in both blood and pulse pressures after injection. However, the secondary effect of YGGFMRFamide, a prolonged reduction in blood pressure, was not produced by FMRFamide. These results suggest that the initial excitatory cardiovascular responses may be due to the presence of the C-terminal amide. All of the cardiovascular effects of injecting these peptides into the lateral ventricle were abolished by pre-treatment with naloxone in a dose that, itself, produced no cardiovascular changes. In conclusion, these peptides seem to act via the naloxone sensitive opiate receptors in the rat brain.

Cold acclimation increases physiological responsiveness to intraventricular injection of beta-endorphin in pentobarbital anaesthetized rats. II. Metabolic function.

beta-endorphin administered intraventricularly into pentobarbital anaesthetized rats led to a reduction in rectal temperature and a dose dependent biphasic response in metabolic rate with increase at low doses (7-10 nmol/kg) and decrease at high doses (30-300 nmol/kg) in rats acclimated to 22 degrees. These metabolic responses to beta-endorphin were enhanced both in magnitude and in duration in rats acclimated to 5 degrees for 3 weeks, indicating an increased responsiveness to beta-endorphin. Naloxone antagonized the metabolic effects of beta-endorphin in rats acclimated to both 22 degrees and 5 degrees, indicating the metabolic effects of beta-endorphin were via the naloxone sensitive opiate receptors.

Central cardiovascular actions of D-Ala2-Met5-enkephalinamide in the rat: effects of naloxone and nucleus reticularis gigantocellularis lesion.

In pentobarbital-anesthetized rats, intraventricular administration of D-Ala2-Met5-enkephalinamide (D-Ala, 30, 100 or 300 nmol/kg) dose-dependently elicited a reduction in arterial pressure. This D-Ala-promoted hypotension was significantly antagonized by naloxone pretreatment (3 mg/kg, i.c.v.) and attenuated by bilateral focal nucleus reticularis gigantocellularis (NRGC) lesions. At higher doses, D-Ala also produced a delayed hypertension that was not only unaffected by naloxone or NRGC lesions, but was in fact potentiated by such pretreatments. We speculate that D-Ala may produce its cardiovascular effect by activating separate subclasses of opiate receptors, possibly at different neural substrates that include the NRGC.

Cold acclimation increases physiological responsiveness to intraventricular injection of beta-endorphin in pentobarbital anaesthetized rats. 1. Cardiovascular functions.

Intraventricular injection of beta-endorphin (3, 7, 10 and 30 nmol/kg) into the third ventricular of pentobarbital-anaesthetized male Sprague-Dawley rats resulted in a dose-dependent increase in mean arterial pressure (MAP) while injection of the same volume of 0.9% NaCl solution did not cause significant changes in MAP. Naloxone, which did not produce any significant change in MAP, antagonized the vasopressor effect of beta-endorphin, indicating that the response is mediated via the naloxone sensitive opiate receptors. Rats acclimated to cold (5 degrees) for 3 weeks showed a potentiated and prolonged increase in MAP following beta-endorphin injection, indicating an increased responsiveness to the peptide. This increased responsiveness in the cardiovascular system is probably of adaptive value in cold acclimation. Naloxone itself did not alter MAP either, but abolished the cardiovascular response to beta-endorphin completely in cold acclimated rats, indicating an increased effectiveness in its antagonistic effect following cold acclimation as well.