A sensitive and efficient LC-MS/MS method for the bioanalysis of fosinopril diacid from human plasma and its application for a bioequivalence study in humans.

Fosinopril diacid is an angiotensin converting enzyme inhibitor with efficient antihypertensive action. It is an active metabolic product formed in the body from hydrolysis of its prodrug Fosinopril. A sensitive, rapid method with high recovery for Fosinopril diacid from human plasma was developed. Solid-phase extraction technique employing Waters Oasis SPE cartridges gave clean samples with very high recovery of 97%. The analyte along with its internal standard (Benazepril hydrochloride) were chromatographed on an XTerra RP8 column (4.6 × 50 mm, 5 µm) using methanol-ammonium acetate buffer (10 mm; 90:10, v/v) as the mobile phase. A triple quadrupole mass spectrometer equipped with electrospray ionization source operated in the negative ion mode was used for detection. Multiple reaction monitoring scan mode was used for monitoring the transitions from m/z 434.00 → 237.15 for Fosinopril diacid and m/z 423.10 → 174.00 for Benazepril hydrochloride. Beer-Lambert's law was obeyed in the range of 0.50-1,500.00 ng/ml (r = 0.9993). The stability of the drugs in human plasma and in stock solution was proved by performing stability tests as per US Food and Drug Administration guidelines. The method was successfully applied for a bioequivalence study of Fosinopril diacid in 36 healthy, adult, male volunteers under fasting conditions.

Crystal structures of angiotensin-converting enzyme from Anopheles gambiae in its native form and with a bound inhibitor.

The mosquitoes of the Anopheles and Aedes genus are some of the most deadly insects to humans because of their effectiveness as vectors of malaria and a range of arboviruses, including yellow fever, dengue, chikungunya, West Nile and Zika. The use of insecticides from different chemical classes is a key component of the integrated strategy against An. gambiae and Ae. aegypti, but the problem of insecticide resistance means that new compounds with different modes of action are urgently needed to replace chemicals that fail to control resistant mosquito populations. We have previously shown that feeding inhibitors of peptidyl dipeptidase A to both An. gambiae and Ae. aegypti mosquito larvae lead to stunted growth and mortality. However, these compounds were designed to inhibit the mammalian form of the enzyme (angiotensin-converting enzyme, ACE) and hence can have lower potency and lack selectivity as inhibitors of the insect peptidase. Thus, for the development of inhibitors of practical value in killing mosquito larvae, it is important to design new compounds that are both potent and highly selective. Here, we report the first structures of AnoACE2 from An. gambiae in its native form and with a bound human ACE inhibitor fosinoprilat. A comparison of these structures with human ACE (sACE) and an insect ACE homologue from Drosophila melanogaster (AnCE) revealed that the AnoACE2 structure is more similar to AnCE. In addition, important elements that differ in these structures provide information that could potentially be utilised in the design of chemical leads for selective mosquitocide development.

The toxicity of angiotensin converting enzyme inhibitors to larvae of the disease vectors Aedes aegypti and Anopheles gambiae.

The control of mosquitoes is threatened by the appearance of insecticide resistance and therefore new control chemicals are urgently required. Here we show that inhibitors of mosquito peptidyl dipeptidase, a peptidase related to mammalian angiotensin-converting enzyme (ACE), are insecticidal to larvae of the mosquitoes, Aedes aegypti and Anopheles gambiae. ACE inhibitors (captopril, fosinopril and fosinoprilat) and two peptides (trypsin-modulating oostatic factor/TMOF and a bradykinin-potentiating peptide, BPP-12b) were all inhibitors of the larval ACE activity of both mosquitoes. Two inhibitors, captopril and fosinopril (a pro-drug ester of fosinoprilat), were tested for larvicidal activity. Within 24 h captopril had killed >90% of the early instars of both species with 3rd instars showing greater resistance. Mortality was also high within 24 h of exposure of 1st, 2nd and 3rd instars of An. gambiae to fosinopril. Fosinopril was also toxic to Ae. aegypti larvae, although the 1st instars appeared to be less susceptible to this pro-drug even after 72 h exposure. Homology models of the larval An. gambiae ACE proteins (AnoACE2 and AnoACE3) reveal structural differences compared to human ACE, suggesting that structure-based drug design offers a fruitful approach to the development of selective inhibitors of mosquito ACE enzymes as novel larvicides.

Simultaneous quantification of fosinopril and its active metabolite fosinoprilat in rat plasma by UFLC-MS/MS: Application of formic acid in the stabilization of an ester-containing drug.

Fosinopril is an angiotensin-converting enzyme inhibitor containing a phosphate ester group which undergoes esterase hydrolysis to its active metabolite, fosinoprilat. EDTA was utilized as an anticoagulant to inhibit the hydrolysis of fosinopril in whole blood during blood collection and processing. To prevent the ex vivo conversion to fosinoprilat, formic acid was added to rat plasma to effectively stabilize fosinopril. A sensitive, rapid and robust ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for simultaneous determination of fosinopril and fosinoprilat in rat plasma. Protein precipitation was employed for plasma sample clean-up. Chromatographic separation was achieved on a Welch Ultimate XB-C18 column using gradient elution with a total run time of 5min. Analytes and their stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometric assay. The assay involves quantitation of both analytes in small-volume (50µL) plasma, with the lower limit of quantification of 0.1 and 1ng/mL for fosinopril and fosinoprilat, respectively. The method was fully validated in linear calibration ranges of 0.1-150ng/mL for fosinopril and 1-1500ng/mL for fosinoprilat with acceptable accuracy and precision. Assay recoveries were high (>95% for fosinopril and >91% for fosinoprilat) and matrix effect was negligible. Both analytes were found to be stable in stabilized rat plasma for 6h at room temperature, 30 days at -80°C, and following three freeze-thaw cycles and were also stable in processed samples for 36h at 4°C. The validated method was successfully applied to sample analyses for pharmacokinetic study of fosinopril and can be extended to the measurement of fosinopril in other biological samples.

Fosinoprilat alleviates lipopolysaccharide (LPS)-induced inflammation by inhibiting TLR4/NF-κB signaling in monocytes.

OBJECTIVE: To evaluate the effect of the fosinoprilat on lipopolysacharides (LPS) induced inflammation in monocytes in vitro. METHODS: Human mononuclear THP1 cells were cultured in complete medium, treated with or without LPS and different concentrations (0,0.25,0.5,1,5,and 10µmol/L) of fosinoprilat. Toll-like receptor (TLR4) mRNA expression was detected by real-time RT-PCR and TLR4 protein level on the surface of monocyte was determined by flow cytometry. Nuclear factor-kappa B (NF-κB) protein level was detected by Western blotting. Cultured supernatant of the THP1 cells in different groups were analyzed by ELISA to detect the levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF-α). RESULTS: Both the mRNA and surface protein level of the TLR4 in the THP1 cells were enhanced by the LPS treatment and down-regulated by pretreatment of the fosinoprilat. Accordingly, LPS-induced NF-κB protein was decreased by the fosinoprilat treatment. The increasing secretion of IL-1ß, IL-6 and TNF-α induced by LPS could also be attenuated by the fosinoprilat treatment. CONCLUSION: The inhibitory effect of the fosinoprilat on the TRL4/NF-κB signaling pathway reveals a potential anti-inflammatory and anti-atherosclerosis target.

Microemulsion liquid chromatographic method for characterisation of fosinopril sodium and fosinoprilat separation with chemometrical support.

The properties of the eluent are the essential factors governing the efficiency in the high-performance liquid chromatography (HPLC) method. A novel approach in retention modelling in the liquid chromatographic separation of fosinopril sodium and its degradation product, fosinoprilat, applying a microemulsion as the mobile phase, was used. The modifications of the mobile phase included the changes to the type of the lipophilic phase, the type and concentration of co-surfactant and surfactant, as well as the pH of the mobile phase. In this study, a full factorial 2(3) design, as the optimal method for screening of the experiment, was applied for selecting factors which had an influence on separation. Optimisation was done by a central composite design. An appropriate resolution with reasonable retention times was obtained with a microemulsion containing 0.9% w/w of cyclohexane, 2.2% w/w of sodium dodecyl sulphate (SDS), 8.0% w/w of n-butanol and 88.9% of aqueous 25 mM disodium phosphate, the pH of which was adjusted to 2.8 with 85% orthophosphoric acid. Separations were performed on an X-Terra 50-mmx4.6-mm, 3.5-mum particle size column at 30 degrees C. UV detection was performed at 220 nm and with a flow rate of 0.3 mL min(-1). The established method was validated and applied for analysis of appropriate tablets. The proposed chromatographic procedure for the separation of fosinopril sodium and its degradation product is less expensive compared with the conventional reversed-phase HPLC method, as well as being simple and rapid. The optimised and validated method can be used for separation, identification and simultaneous determination of fosinopril sodium and fosinoprilat in bulk drug and in pharmaceutical dose forms.

Development of liquid chromatographic method for fosinoprilat determination in human plasma using microemulsion as eluent.

Fosinopril sodium presents a prodrug for the active angiotensin converting enzyme (ACE) inhibitor, fosinoprilat. The dual elimination of fosinoprilat by the liver and the kidney distinguishes fosinopril from other angiotensin converting enzyme inhibitors. Such ways of elimination are important for antihypertensive therapy of patients on haemodialysis. The paper presents development and evaluation of a new and sensitive liquid chromatographic (LC) method for the analysis of fosinoprilat in plasma obtained from patients on haemodialysis. A microemulsion system mixture as mobile phase has been used for the separation and analysis of fosinoprilat in plasma samples. The plasma samples were injected directly onto the HPLC system (Waters Breeze) after appropriate sample dilution with mobile phase. Separations were performed on the Bakerbond ENV 4.6 mm x 150 mm, 5 microm particle size column with UV detection at 220 nm. The flow rate was 1.00 mL min(-1). The mobile phase consisted of 1.0% (w/v) of diisopropyl ether, 2.0% (w/v) of sodium dodecyl sulphate (SDS), 6.0% (w/v) of n-propanol and 91% (w/v) of aqueous 25 mM di-sodium hydrogen phosphate, pH adjusted to 2.8 with 85% orthophosphoric acid. The developed method was then subjected to method validation according to the criteria stated in the FDA bioanalytical method validation guidance. The results for specificity, linearity, low limit of quantification (LLOQ), precision, accuracy and stability were within the accepted criteria. The unique approach applied in this paper makes possible the determination of fosinoprilat even in the presence of metabolites of other drugs, so the method can be used for obtaining the reliable results in a fast and simple way.

Angiotensin converting enzyme inhibition improves cardiac neuronal uptake of noradrenaline in spontaneously hypertensive rats.

OBJECTIVES: It has been shown that a diminished sympathetic activity contributes to the hypotensive and cardioprotective actions of angiotensin converting enzyme (ACE) inhibitors (ACEI). Besides an inhibition of central sympathetic tone and peripheral noradrenaline release, we hypothesized that the interactions of ACEI with the sympathetic system may include a modulation of neuronal catecholamine uptake by peripheral nerves. DESIGN: We investigated the influence of fosinopril on noradrenergic uptake into cardiac neurones in vitro and in vivo in acute and chronic models. METHODS AND RESULTS: Acute administration of fosinoprilat to isolated perfused rat hearts increased the extraction of [3H]-noradrenaline from the perfusate by 39%. Treatment (14 days) of spontaneously hypertensive rats (SHR) with fosinopril (20 mg/kg per day) enhanced the cardiac uptake of i.v. administered [3H]-noradrenaline by 28%. The endogenous left ventricular content of noradrenaline was increased by 49% after an antihypertensive treatment of SHR with fosinopril (20 mg/kg per day). Identical increases in cardiac noradrenaline stores (53%) were observed in SHR treated with a blood pressure ineffective dose of fosinopril (0.2 mg/kg per day). The myocardial content of adrenaline was increased in parallel to noradrenaline after both dose regimes. CONCLUSIONS: It is concluded that ACEI increases neuronal uptake of catecholamines in SHR in a blood pressure-independent manner. This effect occurs acutely and is independent of central sympathetic activity. Therefore, we hypothesize that ACEI modulate the activity of the cardiac noradrenaline transporter by direct activation. The improved uptake of noradrenaline may contribute to the antihypertensive and cardioprotective effects of ACEI.

Endogenous natriuretic peptides participate in renal and humoral actions of acute vasopeptidase inhibition in experimental mild heart failure.

Mild heart failure is characterized by increases in atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in the absence of activation of the renin-angiotensin-aldosterone system (RAAS). Vasopeptidase (VP) inhibitors are novel molecules that coinhibit neutral endopeptidase 24.11, which degrades the natriuretic peptides (NPs) and ACE. In a well-characterized canine model of mild heart failure produced by ventricular pacing at 180 bpm for 10 days, we defined the renal and humoral actions of acute VP inhibition with omapatrilat (OMA, n=6) and acute ACE inhibition (n=5) alone with fosinoprilat. We also sought to determine whether the NPs participate in the renal actions of acute VP inhibition by the administration of OMA together with an intrarenal administration of the NP receptor antagonist HS-142-1 (n=5). OMA resulted in a greater natriuretic response than did ACE inhibition in association with increases in plasma cGMP, ANP, BNP, urinary cGMP, urinary ANP excretion, and glomerular filtration rate (P<0.05 for OMA versus ACE inhibition). Plasma renin activity was increased only in the group subjected to ACE inhibition. Administration of intrarenal HS-142-1 attenuated the renal properties of OMA in association with a decrease in urinary cGMP excretion despite similar increases in plasma ANP and BNP. This study provides new insight into a unique new pharmacological agent that has beneficial renal actions in experimental mild heart failure beyond the actions that are observed with ACE inhibition alone and that are linked to the NP system.

Liquid chromatography/electrospray tandem mass spectrometry method for the quantitation of fosinoprilat in human serum using automated 96-well solid-phase extraction for sample preparation.

A sensitive, specific, accurate and reproducible liquid chromatography/electrospray tandem mass spectrometry method was developed and validated for the quantitation of fosinoprilat in 0.2 mL of human serum. The method employed acidification (with pH 4.0 sodium acetate buffer) of the serum samples to minimize the hydrolysis of the prodrug fosinopril to fosinoprilat prior to purification by automated 96-well solid-phase extraction. The required chromatographic separation of fosinoprilat and fosinopril was achieved isocratically on a Luna C8 analytical column (2 x 50 mm, 3 microm). The total run time was 2 min. The mobile phase contained methanol and water with 10 mM ammonium acetate. Detection was by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 2.00 to 500 ng/mL, was fitted to a 1/x(2) weighted linear regression model. Fosinoprilat quality control (QC) samples used to determine the accuracy and precision of the method were prepared in human serum at concentrations of 5.00, 200, 400 and 1000 ng/mL. The assay accuracy was within 8% (dev). The intra- and inter-assay precisions were within 6 and 3% (RSD), respectively. Fosinopril QC samples used to gauge the rate of hydrolysis of fosinopril to fosinoprilat during the assay procedure were prepared in human serum at 500 ng/mL. The hydrolysis of fosinopril to fosinoprilat was

Liquid chromatographic-electrospray tandem mass spectrometric method for the simultaneous quantitation of the prodrug fosinopril and the active drug fosinoprilat in human serum.

A sensitive, specific, accurate and reproducible LC-MS-MS method was developed and validated for the simultaneous quantitation of the prodrug fosinopril and its active drug fosinoprilat in human serum. The method employed acidification of the serum samples to minimize the hydrolysis of fosinopril to fosinoprilat prior to purification by solid-phase extraction to isolate the two analytes and the two internal standards from human serum. The extracted samples were analyzed by turbo ionspray LC-MS-MS in the positive ion mode. Chromatography was performed on a polymer-based C18 column (Asahipak ODP PVA-C18, 2x50 mm) using gradient elution with methanol and 10 mM ammonium acetate, pH 5.5. The calibration curve, 1.17 to 300 ng/ml, was fitted to a weighted (1/x) linear regression model. Serum quality control (QC) samples used to gauge the accuracy and precision of the method were prepared at concentrations of 5.00, 100, 250 and 500 ng/ml of each analyte. The inter-assay accuracies were within 6% (DEV) for both analytes. The intra- and inter-assay precisions were within 7% and 11% (RSD), respectively, for both analytes. The hydrolysis of fosinopril to fosinoprilat during sample processing was < or = 6%. This degree of conversion would cause little error in the analysis of post-dose serum samples since such samples are known to contain low levels of the prodrug compared to the drug.

Comparison of the pharmacokinetics of fosinoprilat with enalaprilat and lisinopril in patients with congestive heart failure and chronic renal insufficiency.

AIMS: To compare the serum pharmacokinetics of fosinoprilat with enalaprilat and lisinopril after 1 and 10 days of dosing with fosinopril, enalapril and lisinopril. METHODS: Patients with congestive heart failure (CHF, NYHA Class II-IV) and chronic renal insufficiency (creatinine clearance

Pharmacokinetics of fosinoprilat in Chinese and whites after intravenous administration.

The pharmacokinetics of fosinoprilat was studied in 12 healthy Chinese men after a 7.5 mg intravenous dose of fosinoprilat. The data were compared with those from an earlier study using the same protocol in nine healthy white men. Blood and urine samples were obtained before and at various time intervals after fosinoprilat administration up to 24 hours and 48 hours, respectively. Pharmacokinetic parameters were calculated by fitting the plasma or serum concentrations to a three-compartment model. The total clearance (Clt), renal clearance (ClT), and nonrenal clearance (ClNR) were significantly lower in Chinese (16.29 +/- 6.92, 6.85 +/- 2.97, and 9.44 +/- 5.08 mL.hr-1.kg-1) than those obtained in whites (29.88 +/- 6.36, 13.55 +/- 3.45, and 16.33 +/- 5.07 mL.hr-1.kg-1). The Chinese subjects had a significantly lower volume of distribution (Vc [volume of distribution of central compartment] and Vdss [volume of distribution at steady state]) (29.38 +/- 21.12 and 73.67 +/- 40.20 mL/kg) than white men (58.14 +/- 15.01 and 152.49 +/- 24.89 mL/kg). The Chinese men also had a shorter elimination half-life than whites, although not statistically significant. The respective half-lives in Chinese and whites were 5.51 +/- 1.53 and 8.24 +/- 1.99 hours. The significant differences in ClNR and ClR may be related to lower liver elimination function and lower kidney excretory function, respectively. Plasma protein binding may contribute to part of the difference in the volume of distribution. Chinese men have smaller volume of distribution and clearances of fosinoprilat after intravenous administration compared with white men. The cumulative urine excretion of fosinoprilat was not different between Chinese and whites. Chinese may require a lower fosinoprilat dosage to obtain plasma concentrations similar to whites after intravenous administration. However, since a relatively high variation was found in fosinopril oral absorption, the oral dosage of fosinopril for Chinese and whites may not be different. Further study is obviously needed to elucidate whether the pharmacodynamic effect may be different between Chinese and whites.

Fosinopril: pharmacokinetics and pharmacodynamics in congestive heart failure.

Fosinoprilat, the active product of fosinopril, is eliminated by a hepatic pathway, in addition to the renal pathway shared by other angiotensin converting enzyme inhibitors. Congestive heart failure (CHF) may elevate drug plasma concentrations caused by a reduction in steady-state volume of distribution (Vss) and/or an impairment of clearance. This study compared the pharmacokinetics and pharmacodynamics of fosinopril (intravenous and oral) in 10 patients with established CHF and 10 age-, sex-, and weight-matched normal control subjects. There were no statistically significant differences between the patients with CHF and the control patients with respect to maximum drug concentration (Cmax) or area under the plasma concentration-time curve from 0 to infinity. Absolute bioavailability was approximately 29%. Vss was similar, and protein binding was 99% in both groups. The oral half-life of fosinoprilat was significantly longer than the intravenous half-life for both the patients with CHF and normal subjects, without statistically significant differences between the study groups. Median time to reach Cmax occurred at 4 hours in each group and corresponded to maximum angiotensin converting enzyme inhibition, which was essentially complete through 12 hours and markedly reduced through 24 hours. Thus these data indicate that patients with CHF can receive fosinopril without undue increases in fosinoprilat concentrations. This probably is due to the dual excretory pathways.

Effects of metalloproteinase inhibitors on leukotriene A4 hydrolase in human airway epithelial cells.

Human neutrophil leukotriene A4 (LTA4) hydrolase is a zinc-containing metalloproteinase with aminopeptidase activity and can be inhibited by some metalloproteinase inhibitors. Human airway epithelial cells also contain an LTA4 hydrolase enzyme that has some novel properties, suggesting that this enzyme may be functionally and structurally unique. Thus, we questioned whether the epithelial cells were studied either intact or disrupted. Of the metalloproteinase inhibitors examined, only captopril, bestatin, and fosinoprilat had appreciable inhibitory activity for LTA4 hydrolase in disrupted epithelial cells. Concentration-inhibition curves to captopril, bestatin, and fosinoprilat revealed IC50 values of 430 microM, 7 microM, and 1 mM, respectively, for disrupted-cell LTA4 hydrolase activity. In contrast to its effects on neutrophils, 1,10-O-phenanthroline had no significant effect on disrupted epithelial cell hydrolase activity and had only minimal effects when this activity was partially purified (179-fold). LTA4 hydrolase concentration-inhibition curves examined in intact cells with captopril, bestatin, and 1,10-O-phenanthroline revealed IC50 values of 63, 70, and 920 microM, respectively. Aminopeptidase activity in disrupted epithelial cells was inhibited by amastatin, bestatin, and 1,10-O-phenanthroline (IC50 values of 500 nM, 1 microM, and 17 microM, respectively), but not by captopril at the highest concentration tested, 10 mM. These findings are in contrast to prior studies in neutrophils. When neutrophils were stimulated with A23187 after treatment with captopril, transcellular synthesis of LTB4 was inhibited more effectively than direct synthesis of leukotriene B4 (LTB4) (43.8 +/- 2.5 vs 18.5 +/- 4.7%; N = 8, P < 0.02). We conclude that LTA4 hydrolase activity of human airway epithelial cells is inhibited by some metalloproteinase inhibitors, but that the profile of inhibition is distinct from that for the neutrophil enzyme. These data provide additional information that LTA4 hydrolase in the epithelial cell is a novel enzyme, distinct from that found in the neutrophil.

Single-dose and steady-state pharmacokinetics of fosinopril and fosinoprilat in patients with hepatic impairment.

The single-dose and steady-state pharmacokinetics of the angiotensin-converting enzyme (ACE) inhibitor fosinopril and its active diacid, fosinoprilat, were evaluated in 6 healthy volunteers and 12 patients with alcoholic cirrhosis. Fosinopril was administered at a dosage of 10 mg once daily for 14 days. Results in the two groups were similar, with no evidence of accumulation of fosinoprilat in hepatically impaired patients. Mean (+/- SD) maximum observed plasma concentrations of fosinoprilat in the healthy subjects were 112.0 +/- 67.2 ng/mL after the first dose and 144.1 +/- 61.7 ng/mL at steady-state. Corresponding values for the hepatically impaired patients were 111.4 +/- 40.1 ng/mL and 140.2 +/- 50.9 ng/mL. The area under the serum concentration versus time curve for healthy volunteers was 790.7 +/- 431.0 ng.hr/mL after the first dose and 940.3 +/- 400.4 ng.hr/mL at steady-state. Similar values were noted in hepatically impaired patients: 926.0 +/- 293.9 ng.hr/mL and 1,255.4 +/- 434.0 ng.hr/mL for first dose and steady-state, respectively. No statistically significant differences were detected in fosinoprilat pharmacokinetic values between healthy and hepatically impaired subjects. Absence of accumulation can be attributed to the dual route of elimination of fosinoprilat reported in previous studies. Renal excretion of fosinoprilat in hepatically impaired patients prevents increased accumulation. The present findings suggest that the starting dose of fosinopril used in hypertensive patients with normal renal and hepatic function can also be used in patients with hepatic impairment secondary to cirrhosis.

Fosinoprilate prolongs the action potential: reduction of iK and enhancement of the L-type calcium current in guinea pig ventricular myocytes.

OBJECTIVE: The aim was to study the effect of fosinoprilate, a new ACE inhibitor, on the action potential and plateau currents of cardiac muscle. METHODS: Whole cell patch technique was used to record action potentials (n = 6), the L-type iCa (iCaL; n = 5), in some cases (n = 4) also using Cs+ loaded pipettes; with 5 mM Co2+, the time dependent K+ current (IK) underlying delayed rectification was analysed in guinea pig ventricular myocytes (n = 3). RESULTS: Fosinoprilate prolonged the 50% repolarisation (APD50) from 440(SEM 50) ms to 485(48) ms (0.1 microM), to 525(46) ms (0.3 microM), to 632(58) ms (1 microM), and to 702(69) ms (3.0 microM). The APD90 was delayed from 510(63) ms to 540(45) ms (0.1 microM), to 583(42) ms (0.3 microM), to 702(62) ms (1.0 microM), and to 765(72) ms (3.0 microM). Higher concentrations (10-100 microM) caused early afterdepolarisations, very long action potentials, and irregular oscillations. ICaL was enhanced by up to 183%, showing a Kd of 0.2 microM; in contrast to the steady state activation (d infinity), the inactivation curve f infinity was shifted in the depolarising direction, considerably enlarging the Ca2+ window. Slow inactivation time course was unchanged, whereas the fast time constant (tau f) was accelerated. Fosinoprilate reduced the outward current during depolarising clamps from 1.7(0.2) nA to 1.41(0.11) nA with a 0.1 microM dose, and to 0.54(0.14) nA with a 1.0 microM dose; the tails were decreased from 0.39(0.03) nA to 0.27(0.03) nA with 0.1 microM and to 0.13(0.02) nA with 1.0 microM. Kinetics of IK were unaltered. Computer simulations based on these data using the OXSOFT-HEART program mimicked the results rather closely. CONCLUSIONS: The results suggest that fosinoprilate prolongs the plateau due to a partial block of iK and an extension of the Ca2+ window by 10 mV, causing a class III antiarrhythmic effect. High concentrations further open the Ca2+ window resulting in early afterdepolarisations and plateau oscillations and may cause an inward transport of Ca2+ ions by the Na-Ca exchange.

The pharmacokinetics and pharmacodynamics of fosinopril in haemodialysis patients.

The pharmacokinetics and pharmacodynamics of fosinoprilat, the diacid of fosinopril sodium (a new angiotensin-converting enzyme (ACE) inhibitor), were investigated in six haemodialysis patients. Intravenous 14C-fosinoprilat (7.5 mg), oral 14C-fosinopril sodium (10 mg) and oral fosinopril sodium (10 mg) were administered in an open-label, randomized study. Mean maximum concentration (Cmax), clearance (CL), volume of distribution at steady-state (Vss), mean residence time (MRTiv), and t1/2 values after IV administration of 14C-fosinoprilat were 2,042 micrograms.ml-1, 11.3 ml.min-1, 11.0 l, 16.3 h and 28.3 h, respectively. Following oral administration of 14C-fosinopril, mean Cmax, time to maximum plasma concentration (tmax), and fosinoprilat bioavailability values were 197 ng.ml-1, 5.2 h and 29.2%. Para-hydroxy fosinoprilat and fosinoprilat glucuronide comprised approximately 15% and 2% of radioactivity recovered in faeces. Four hours of haemodialysis only cleared approximately 1.5% of the administered dose. The maximum effect (Emax) model was fitted to the percentage inhibition of serum ACE activity vs. fosinoprilat concentration data in three patients. Emax ranged from 95.3 to 102.5%, and IC50 (the fosinoprilat concentration required to produce 50% of Emax) ranged from 2.6 to 4.2 ng.ml-1. Pharmacokinetic variables of the patients were similar to those in patients with moderate to severe renal dysfunction. Dosage modifications or supplemental dosing following dialysis are unnecessary.

Local potentiation of bradykinin-induced vasodilation by converting-enzyme inhibition in isolated coronary arteries.

The interaction of angiotensin-converting enzyme (ACE) inhibitors and bradykinin was investigated in isolated bovine and human coronary arteries. Rings with and without endothelium were mounted in organ chambers for measurement of isometric force. The effects of the ACE inhibitors lisinopril, enalaprilat, fosinoprilat, ramiprilat, and captopril were determined during submaximal stimulation with bradykinin or other vasodilators. Lisinopril and captopril alone did not affect vascular tone; however, in rings with endothelium partially relaxed with bradykinin (> or = 10(-10) M), all ACE inhibitors caused further relaxations. Lisinopril did not affect bradykinin concentrations in the incubation medium. Mechanical removal of the endothelium or incubation with nitro-L-arginine or the bradykinin2-receptor antagonist Hoe 140 prevented the relaxations to bradykinin and lisinopril. Other vasodilators including acetylcholine, adenosine diphosphate, substance P, or SIN-1 did not prime the rings to respond to ACE inhibitors. Endothelium-dependent relaxations to lisinopril were also observed in human coronary arteries treated with bradykinin (> or = 10(-7) M). Thus, ACE inhibitors potentiate endothelium-dependent relaxations to submaximal concentrations of bradykinin in bovine and human coronary arteries. This local mechanism occurs regardless of elevated bradykinin concentrations in the blood and reduced angiotensin II generation.

Effects of different angiotensin-converting enzyme (ACE) inhibitors on ischemic isolated rat hearts: relationship between cardiac ACE inhibition and cardioprotection.

We determined the relationship between cardiac angiotensin-converting enzyme (ACE) inhibition and anti-ischemic efficacy of several structurally different ACE inhibitors or their prodrug esters perfused through the isolated rat heart. Seven ACE inhibitors inhibited cardiac ACE to varying degrees due to differences in uptake during perfusion through nonischemic rat hearts. Zofenopril-sulfhydryl and fosinoprilic acid were the most effective of the free inhibitors. Among the prodrugs, zofenopril and S-benzoylcaptopril, hydrolyzed rapidly by cardiac esterase, were more effective than their component ACE-inhibitors, whereas fosinopril, ramipril and enalapril were poorly active. For studies in ischemic rat hearts, vehicle or drug treatment was initiated 10 min before a 25-min period of global ischemia and during a 30-min reperfusion period. Of five unesterified ACE inhibitors studied for anti-ischemic activity, only captopril and zofenopril-sulfhydryl were found to improve postischemic contractile function and reduce cell death in the isolated rat hearts. Fosinoprilic acid, ramiprilat and enalaprilat were not cardioprotective at high perfusion concentrations, despite the fact that nearly complete inhibition of cardiac ACE was achieved with all of the compounds studied. The S-benzoyl prodrugs of zofenopril-sulfhydryl and captopril were at least as potent as their component ACE inhibitors in reducing ischemic-reperfusion damage in the same model. Neither zofenopril nor captopril, however, had any effect on coronary flow before or after ischemia. Thus, it appears that the cardioprotective effects of zofenopril and captopril are independent of cardiac ACE inhibition or, at least, that ACE inhibition alone is not sufficient. Both captopril and zofenopril are sulfhydryl-containing compounds whereas the inactive compounds are not; and, thus, this group appears to be important in mediating their cardioprotective actions.