Rapid determination of sildenafil and its analogues in dietary supplements using gas chromatography-triple quadrupole mass spectrometry.

Application of gas chromatography-triple quadrupole mass spectrometry for identification, confirmation and quantification of 6 phosphodiesterase-5 (PDE-5) inhibitors (sildenafil, dimethylsildenafil, homosildenafil, thiosildenafil, thiodimethylsildenafil and thiohomosildenafil) in dietary supplements was investigated. The MS was operated in multiple reaction monitoring mode, for better sensitivity and selectivity. In this manner, the method is adequate to reduce background noise with less interference from co-eluting compounds in the samples. Two different ionisation techniques, electron ionisation (EI) and chemical ionisation (CI), were studied and compared. The chromatographic separation was performed on a short 10 m non-polar capillary column without any derivatisation step. This permitted fast analysis for all analogues with retention time less than 11 min, for both techniques. Use of backflushing can aid method retention time reduction and improves column maintenance. Evaluation of method validation included limit of detection (LOD), lower limit of quantitation (LLOQ), linearity, precision and recovery were performed for both EI and CI techniques. The LOD obtained varied from 0.03 to 1.50 µg/g and the LLOQ ranged from 0.10 to 5.00 µg/g. Good calibration linearity was obtained for all analogues for both techniques, with correlation coefficients (r(2)) higher than 0.99. Mean recoveries of all analogues using CI show higher values (83.4-108.8%) than that of EI (61.9-91.1%). The intra- and inter-assay precisions were evaluated for all analogues at spiked concentration of 10 µg/g and the relative standard deviation was less than 15% for both methods. These methods were then successfully applied to dietary supplement samples without prior derivatisation, confirming that the samples were adulterated with sildenafil and/or its analogues.

Clopamide: plasma concentrations and diuretic effect in humans.

Clopamide pharmacokinetics were determined after oral doses of 5, 10, and 20 mg in normal volunteers. Maximum plasma concentrations occurred within 2 hours and were followed by a monoexponential decline with an elimination half-life of approximately 10 hours. There was an approximately linear relationship between dose and the AUC. Urinary sodium, chloride, and potassium excretion rates indicated that the peak diuretic activity corresponded with peak plasma drug concentrations and probably continued for 12 to 24 hours. There was little difference between the total sodium and chloride output after each dose of clopamide, suggesting that 5 mg may have been close to the top of the dose-response curve. Chlorothiazide, 500 mg, caused less sodium and chloride output with similar potassium loss. During chronic administration to patients with hypertension, hypokalemia was more marked with clopamide, 10 mg daily, than with clopamide, 5 mg, or chlorothiazide, 500 mg daily.

The effect of hepatic cirrhosis on the pharmacokinetics and blood pressure response to nicardipine.

The present study was designed to compare the pharmacokinetic handling of a single oral dose of nicardipine in normal subjects and in patients with hepatic cirrhosis and to compare the sensitivity of the two groups to its hypotensive effect. Nicardipine plasma concentrations were substantially higher in the subjects with hepatic cirrhosis with impaired antipyrine clearance, as shown by a significantly higher average Cmax and AUC. The terminal elimination half-life in this group varied from 0.8 to 60.2 hours (median, 11.7 hours), compared with 0.6 to 4.1 hours (median, 1.4 hours) in the group of eight subjects with normal liver function. In the cirrhotic patients with impaired antipyrine clearance, the AUC of the pyridine metabolite averaged 10% of that of the parent drug, whereas in normal subjects the ratio averaged 48%. This finding suggests less conversion of nicardipine to this metabolite in subjects with impaired hepatic function. Peak blood pressure decreases were greater in the cirrhotic group, which was in keeping with the higher plasma levels in these subjects.

Single-dose and steady-state pharmacokinetics of doxazosin given in combination with chlorothiazide to hypertensive subjects.

The pharmacokinetics of doxazosin were determined in hypertensive subjects after a single dose of 1 mg, and at steady-state while receiving doses of 1, 2, 4 and 8 mg of the drug daily. Chlorothiazide 500 mg once daily was administered as additional therapy throughout the study. After a single dose doxazosin was rapidly absorbed, with peak plasma drug concentrations (Cmax) occurring after 2.1 +/- 0.4 hours. The elimination half-life in plasma was 10.7 +/- 1.2 hours. These parameters remained essentially unchanged during maintenance administration of doxazosin at each of the dose levels. Calculations of Cmax and area under the concentration-time curve (AUC0----infinity) indicated that the pharmacokinetic disposition of the drug remained linear over the dose range 1 to 8 mg.

Beta-adrenoceptor-blocking drugs: current status and the significance of partial agonist activity.

There is some evidence that partial agonism is an important property of beta-blocking drugs, and this property may be exploited to produce a new range of positive inotropic drugs. With the established beta-adrenoceptor-blocking drugs, the level of partial agonist activity is weak and the dose-response curve for this property is shallow. However, its absence appears to increase the likelihood of inducing bronchospasm and bradycardia, and drugs that lack intrinsic sympathomimetic activity appear more likely to be associated with rebound cardiac arrhythmias on cessation of treatment. The idea of a small level of hormone activity--in this case, catecholamine activity--being necessary to maintain normal cardiac and perhaps bronchial function is not new. Minimal doses of steroids are essential to maintaining the inotropic action of cardiac muscle. There is now enough accumulated evidence to suggest that a minimal degree of beta-adrenoceptor stimulation is also important for normal bronchial and cardiac function, and its absence increases the incidence of bradycardia and the risks of bronchospasm and rebound arrhythmias.

S-nitrosothiols as vasodilators: implications regarding tolerance to nitric oxide-containing vasodilators.

1. The formation of an S-nitrosothiol compound, S-nitroso-N-acetylcysteine (SNAC) has recently been proposed to mediate the augmentation of the anti-aggregatory and haemodynamic effects of glyceryl trinitrate observed in the presence of N-acetylcysteine. This study investigated the effects on an isolated coronary artery preparation of acute and prolonged exposure to S-nitrosothiol compounds and nitric oxide (NO). 2. Single doses of NO and of the S-nitrosothiol compounds, SNAC and S-nitroso-N-acetyl-penicillamine (SNAP), induced rapid, but transient, relaxations in U46619-contracted bovine isolated coronary artery rings. Peak relaxation responses to SNAP and NO were attenuated in the presence of N-acetylcysteine, cysteine, ascorbic acid and methylene blue. The duration of the relaxation responses to SNAC was two to three times longer than those to SNAP and NO. In the presence of N-acetylcysteine (but not cysteine, ascorbic acid or methylene blue) the duration of the relaxation responses to SNAP and NO (but not to SNAC) was markedly increased. H.p.l.c. assay confirmed that, in the presence of N-acetylcysteine, SNAP and, to a lesser degree, NO were converted to the relatively more stable and longer acting vasodilator, SNAC. 3. When compared to control rings, coronary artery rings superfused with glyceryl trinitrate were subsequently markedly less responsive to the vasodilator actions of glyceryl trinitrate, whereas responsiveness to SNAC or NO was only marginally reduced. On the other hand, coronary artery rings superfused with SNAC or NO were subsequently less responsive to glyceryl trinitrate, SNAC and NO. Thus prolonged vascular exposure to SNAC or NO induced a form of tolerance different from that induced with glyceryl trinitrate and which is possibly associated with impaired guanylate cyclase activity. 4. Coronary artery rings superfused with NO were markedly less responsive to glyceryl trinitrate and NO, whereas responses to the endothelium-dependent vasodilator A23187 and to theophylline were not significantly attenuated. 5. It is concluded that formation of the more stable vasodilator SNAC occurs on incubation of N-acetylcysteine with SNAP or NO. While coronary artery responsiveness to SNAC and NO is virtually unchanged in the presence of glyceryl trinitrate-induced tolerance, after prolonged exposure to SNAC or NO tolerance may develop to these vasodilators with cross-tolerance to glyceryl trinitrate but not A23187. Thus, formation or therapeutic utilization of SNAC may acutely circumvent the problem of glyceryl trinitrate-induced tolerance but, during prolonged vascular exposure to SNAC, attenuation of vascular responsiveness may occur to a wide range of vasodilators.

Pharmacology of combined alpha-beta-blockade. I.

Several compounds of the chemical class arylethanolamines have been shown to possess combined alpha- or vasodilator and beta-adrenoceptor blocking properties. The first drug was labetalol (AH5158)[5-(1-hydroxy-2)1-methyl-3-phenylpropyl(amino)-ethyl (salicylamide)]. Others include medroxalol, bucindolol and YM-09538, which differ from labetalol either by the nature of the substitution on the primary benzene ring and/or on the terminal nitrogen. All of these drugs are non-selective beta-blockers, except for bucindolol whose selectivity has not been carefully defined. The rationale for the development of this group of drugs was the knowledge that blockade of one adrenoceptor subtype causes reflex stimulation of the other, i.e. vasoconstriction after nonspecific beta-blockade and tachycardia after alpha-blockade. Since both of these compensatory responses act to prevent a fall in blood pressure, a relatively weak blockade of both receptor types should act synergistically to produce a lowering of blood pressure with minimal physiological disturbance. Haemodynamic studies have confirmed that the additional alpha-blocking properties of labetalol produce a pattern of haemodynamic changes unlike that of propranolol and other simple beta-adrenoceptor blocking agents. Peripheral vascular resistance, which falls acutely during the initial administration of the drug, tends to fall further during prolonged administration and the pulse rate tends to remain only slightly lower than pretreatment levels. In addition, at normal dose levels cardiac output is maintained by a compensatory increase in stroke volume. Thus, blood pressure is lowered largely by a reduction in vascular resistance, and although the heart rate falls significantly during exercise, the cardiac output is maintained by an increase in stroke volume. This pattern of events is different to that seen with beta-blocking agents which consistently reduce cardiac output during exercise. Currently labetalol is the only member of this group of drugs which is in established clinical use. Its antihypertensive efficacy has been confirmed in many studies and it has been shown to be effective in the management of both hypertensive emergencies and in the long term management of severe hypertension. It is particularly valuable in allowing a reduction in the number of drugs required for adequate blood pressure control. The early theoretical prediction that postural hypotension would occur with high doses is now acknowledged to be labetalol's major dose-limiting side effect. Most of the available pharmacokinetic data on labetalol were derived from studies which utilised a fluorimetric assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrolysis of angiotensin I by peptidases in homogenates of rat lung and aorta.

The hydrolytic cleavage of angiotensin I has been studied in homogenate preparations of rat lung and aorta using gradient elution HPLC to monitor the formation of peptide products. Fresh crude homogenate preparations produced a rapid breakdown of angiotensin I to largely unidentifiable fragment peptides. Neither His-Leu nor angiotensin II was observed in these preparations even in the presence of captopril (20 microM) and the amino-peptidase inhibitors, puromycin, amastatin and bestatin. However, in freeze-thawed homogenates, angiotensin II and His-Leu were detectable together with the tetrapeptide, angiotensin (1-4). The addition of captopril (20 microM) reduced the amount of angiotensin II produced but did not completely block its formation. Higher concentrations of captopril or the addition of enalaprilat or EDTA did not further reduce the amount of angiotensin II produced. In the presence of captopril a peptide corresponding to des-Leu(10)angiotensin I was formed in relatively large amounts (equivalent to 40% of angiotensin I catabolized). Homogenates purified by concanavalin A affinity chromatography gave a clean hydrolysis of angiotensin I to angiotensin II and His-Leu which was completely blocked by captopril. These results suggest an ACE-like activity in rat lung and aorta that is not sensitive to converting enzyme inhibitors.

Captopril disulfide conjugates may act as prodrugs: disposition of the disulfide dimer of captopril in the rat.

The absorption and metabolism of the disulfide dimer conjugate of captopril has been studied in the rat following both oral and intravenous dosing and compared with that of the active monomer, captopril. Metabolism of the dimer to captopril has been shown after both oral and intravenous administration of the dimer (10 mg/kg) with peak plasma levels of captopril (154 ng/ml) occurring at 1 hr post dose. By contrast the peak plasma level of captopril after oral administration of captopril (10 mg/kg) at the same dose was much higher at 678 ng/ml and also occurred at 1 hr post dose. Plasma captopril disulfide species were much higher than the plasma levels of captopril after the administration of either dimer or captopril and tended to persist for much longer than for monomeric captopril particularly after administration of the dimer. Both the dimer and its pharmacologically active product captopril were found in relatively large amounts in lung, kidney and liver following the oral administration of the dimer.

Formation of angiotensin II and other angiotensin peptides from des-leu 10-angiotensin I in rat lung and kidney.

The formation of AII from a metabolite of AI, des-leu10-angiotensin I [A(1-9)] has been studied in centrifugal fractions of rat lung and kidney using gradient elution HPLC to monitor the formation of peptide products. AII-forming activity was present in kidney S2 (22.3 nmol/mg protein/min) but not in kidney P2 centrifugal fractions. Lung S2 fractions showed relatively weak AII-forming activity (0.34 nmole/mg protein/min) whilst no activity was observed in lung P2. Carboxypeptidase N-like activity measured using both Hipp-Arg and Hipp-Lys as synthetic substrates did not parallel AII-forming activity, since this activity was highest in the P2 fractions of both lung and kidney, as were ACE and aminopeptidase activities. Whilst the major peptide produced in kidney S2 was AII (71%) significant amounts of both AIII (23%) and A(2-9) (6%) were also observed. In lung the amounts of these peptides produced as a percentage of the A(1-9) degrading activity were 2.9%, 2.4% and 21% respectively. The AII-forming activity in kidney S2 was not inhibited by enalaprilat, bestatin, amastatin, phosphoramidon or Pro-Phe but was inhibited (31%) by 1 mM cobalt (II). 1,10-Phenanthroline, iodoacetic acid, EDTA and puromycin significantly enhanced the formation of AII and increased the rate of degradation of the substrate, A(1-9). These results support the concept of a sequential carboxypeptidase pathway operating, particularly in kidney, to produce AII from AI. These results provide further evidence of an alternative metabolic pathway for the formation of AII not involving angiotensin converting enzyme.

Reversibility of disulfide formation. Comparison of chemical and enzyme-mediated reduction of penicillamine and captopril disulfides.

The reduction of penicillamine disulfide by reductants in aqueous solutions has been studied and compared with that for captopril disulfide. Whereas near quantitative reduction for captopril disulfide was achieved with tributyl phosphine (200 mM), no detectable penicillamine was formed from penicillamine disulfide. Thiol reductants (25 mM) were, however, partially able to reduce penicillamine disulfide with the most effective agent being glutathione (15% reduction) following by dithioerythritol (8%) and cysteine (5.1%). The reduction of penicillamine-cysteine disulfide by glutathione was 6-fold higher than for penicillamine disulfide. Kinetic analysis showed that the initial rate of reduction and equilibrium constant for the reduction of penicillamine disulfides by glutathione were 267- and 875-fold less than for captopril disulfide at pH 7.4. Biotransformation studies in the cytosol fraction of rat blood cells demonstrated that whereas 48% of the reduction of captopril disulfide was enzyme-mediated only 19% of the penicillamine formed was enzyme-mediated for penicillamine disulfide. Accumulation of disulfides of penicillamine in patients taking penicillamine may therefore be a problem during chronic therapy.

Effect of chronic enalapril treatment on enzymes responsible for the catabolism of angiotensin I and formation of angiotensin II.

We have investigated the effect of chronic administration of enalapril on the carboxypeptidases responsible for the formation of angiotensin II from angiotensin I and other peptidases known to recognize angiotensin I as a substrate in the rat. These studies have shown an increase in activity in rate of formation of des-Leu-angiotensin I in both kidney S2 and P2 centrifugal fractions as well as a decrease in the rate of degradation of angiotensin I substrate. Similar increases in the formation of A(1-8) have been observed in kidney using A(1-9) as substrate. These two enzyme activities have been named carboxypeptidase K1 and K2, respectively to reflect their presence in rat kidney. These changes were accompanied by significant decreases in the activity of an amastatin-sensitive aminopeptidase and endopeptidase 24.11 in the kidney P2 fraction. These data suggest that chronic treatment with ACE inhibitors may differentially affect the activity of other enzymes capable of degrading angiotensin causing a substantial re-direction of angiotensin metabolism.

Tissue distribution of captopril, reducible captopril conjugates and S-methylcaptopril in the rat.

The tissue distribution of captopril, an antihypertensive drug possessing a free sulfhydryl group, and its sulfur-conjugated metabolites was studied in rats by gas chromatography-mass spectrometry at 15, 30 and 60 min following a single 10 mg/kg oral dose of captopril. It was found that tissue accumulation of captopril was rapid with both free and oxidized-forms already present at 15 min post-dose. A maximum concentration of captopril was achieved at 30 min in tissues studied, being substantially higher in kidney (14.2 micrograms/g), with lesser amounts occurring in liver, lung, heart, blood cells, spleen and plasma in that order. Oxidized disulfide forms of captopril were usually present in the same or slightly higher proportion than free captopril except for liver which only contained detectable disulfides at 15 min after oral dosing. S-methylcaptopril was also present at 30 min in all tissues examined with highest levels occurring in liver and kidney (1.05 micrograms/g) followed by plasma, lung, heart, spleen and blood cells.

S-methylation of captopril. Demonstration of captopril thiol methyltransferase activity in human erythrocytes and enzyme distribution in rat tissues.

The presence of a methyltransferase enzyme in human red blood cells (RBCs) capable of S-methylation of captopril is described. The apparent Michaelis-Menten (Km) constant for captopril was 0.5 mM and the maximum velocity (Vmax) was 0.391 pmoles S-methylcaptopril (mg protein)-1 min-1. There is some evidence presented to show that S-methylcaptopril inhibited its own formation with a ki value of 5.81 mM. Captopril thiol methyltransferase activity was also examined in rat tissues and was found to be present in all tissue studied. Subcellular localisation studies in rat liver suggest that the enzyme was microsomal in origin. The order of activity was liver greater than heart greater than spleen greater than lung greater than kidney much greater than RBC (rat). This tissue distribution was quite different from previous studies using other thiol substrates and is consistent with more than one form of thiol methyltransferase enzyme in tissues.

Effect of probenecid on the disposition of captopril and captopril dimer in the rat.

The urinary excretion of captopril has been studied in a bladder-cannulated rat model and compared with that obtained after co-administration with probenecid. Probenecid reduced significantly the urinary excretion of captopril from 41% to 21% of the administered dose over a 3-hr period and significantly lowered urine flow rates. In addition, the effect of probenecid on plasma levels of captopril and total captopril (captopril plus disulfides) after oral administration of the disulfide prodrug captopril dimer (10 mg/kg) has been studied in a conscious rat preparation. Co-administration of probenecid (20 mg/kg) given either orally or intravenously increased both the plasma levels of captopril and total captopril (captopril plus captopril disulfides) over a 4-hr period. A prolonged significant inhibition of plasma ACE after co-administration of probenecid and captopril dimer suggests that probenecid may be useful to prolong the action of captopril or the prodrug captopril dimer.

Radioimmunoassay for immunoreactive [des-Leu10]-angiotensin I.

A specific radioimmunoassay for the angiotensin-derived peptide [des-Leu10]-angiotensin I (AI-dL) is described. Antisera obtained from rabbits injected with immunogen prepared by coupling bovine beta-thyroglobulin to the peptide with carbodiimide were specific to this peptide and did not recognise related angiotensin peptides such as AI, AII, AIII, nor did they recognise other peptides such as bradykinin, substance P, bombesin or dynorphin(1-8). Immunoreactive AI-dL was detected for the first time in the plasma of rats and humans following purification by HPLC at concentrations of 78 and 40 pg/ml, respectively. Concentrations of AI-dL are increased following chronic administration of captopril to rats.

Bradykinin-potentiating activity of captopril disulphide dimer (SQ 14,551).

The ability of captopril and one of its metabolites, the disulphide dimer have been studied for their ability to affect angiotensin I and bradykinin responses. Captopril disulphide dimer (i.v.) potentiated the vasodilatory effects of bradykinin in urethane-anaesthetized rats, at doses of 0.1 and 0.3 mg/kg but did not inhibit the angiotensin I-mediated pressor response at these same concentrations. This activity of captopril disulphide dimer in vivo was not seen with bradykinin responses in isolated guinea pig ileum except at bath concentrations much higher than for captopril (10(-5) M). However captopril and captopril disulphide dimer at oral doses of 10 mg/kg both lowered systolic and diastolic blood pressures in spontaneously hypertensive rats. These studies are consistent with an in vivo bradykinin-potentiating activity of captopril disulphide dimer and suggest a possible antihypertensive activity of disulphide metabolites of captopril.

Heroin-related deaths in Victoria: a review of cases for 1997 and 1998.

The objectives of this paper were to determine the number of heroin-related deaths in Victoria for the years 1997-1998 and to detail the demography and toxicology findings, and also compare heroin death rates for this decade. The number of deaths attributed to the intravenous use of heroin has increased dramatically in Victoria since 1990. The increases were 5-fold. There were 166 deaths in 1997 and 268 in 1998. The heroin death is typified by a median age of 30 for males and 29 for females, although the age range is from children as young as 15 to adults in their sixth decade of life. Over 85% of cases were using other central nervous system depressants, with benzodiazepines (45%) and alcohol (36%) being the most common. Approximately 60% occurred indoors at a private residence, the remainder occurred in public places and other locations. A similar number (60%) died alone. A wide distribution of deaths occurred throughout the metropolitan and regional areas showing a growing spread in the heroin problem in the community.

Dose related risk of motor vehicle crashes after cannabis use.

The role of Delta(9)-tetrahydrocannabinol (THC) in driver impairment and motor vehicle crashes has traditionally been established in experimental and epidemiological studies. Experimental studies have repeatedly shown that THC impairs cognition, psychomotor function and actual driving performance in a dose related manner. The degree of performance impairment observed in experimental studies after doses up to 300 microg/kg THC were equivalent to the impairing effect of an alcohol dose producing a blood alcohol concentration (BAC) >/=0.05 g/dl, the legal limit for driving under the influence in most European countries. Higher doses of THC, i.e. >300 microg/kg THC have not been systematically studied but can be predicted to produce even larger impairment. Detrimental effects of THC were more prominent in certain driving tasks than others. Highly automated behaviors, such as road tracking control, were more affected by THC as compared to more complex driving tasks requiring conscious control. Epidemiological findings on the role of THC in vehicle crashes have sometimes contrasted findings from experimental research. Case-control studies generally confirmed experimental data, but culpability surveys showed little evidence that crashed drivers who only used cannabis are more likely to cause accidents than drug free drivers. However, most culpability surveys have established cannabis use among crashed drivers by determining the presence of an inactive metabolite of THC in blood or urine that can be detected for days after smoking and can only be taken as evidence for past use of cannabis. Surveys that established recent use of cannabis by directly measuring THC in blood showed that THC positives, particularly at higher doses, are about three to seven times more likely to be responsible for their crash as compared to drivers that had not used drugs or alcohol. Together these epidemiological data suggests that recent use of cannabis may increase crash risk, whereas past use of cannabis does not. Experimental and epidemiological research provided similar findings concerning the combined use of THC and alcohol in traffic. Combined use of THC and alcohol produced severe impairment of cognitive, psychomotor, and actual driving performance in experimental studies and sharply increased the crash risk in epidemiological analyses.

High-performance liquid chromatographic determination of platinum (II) in plasma ultrafiltrate and urine: comparison with a flameless atomic absorption spectrometric method.

A technically simple, rapid and sensitive high performance liquid chromatographic assay for cis-dichlorodiammineplatinum (II) in human plasma ultrafiltrate and urine is described. The drug was chelated by exchange with diethyldithiocarbamate and extracted into chloroform. Nickel (II) was used as an internal standard which allows correction for the matrix effects observed with previous chromatographic and spectrometric methods. Chromatography was performed on a mu-Bondapak CN column and the eluent measured spectrophotometrically at 254 nm. Precision and reproducibility were both excellent and the detection limit was less than 50 ng/ml using only 1 ml of biological fluid.