Population pharmacokinetics of ramipril in patients with chronic heart failure: A real-world longitudinal study.

In patients with chronic heart failure (CHF), the use of angiotensin-converting enzyme inhibitors, including ramipril, is recommended to reduce the risk of heart failure worsening, hospitalisation, and death. Our aim was to investigate the influence of body composition on the pharmacokinetics of ramipril and its active metabolite ramiprilat and to evaluate the changes in pharmacokinetics after prolonged therapy. Twenty-three patients with CHF who were on regular therapy with ramipril participated at the first study visit ( median age 77 years, 65 % male, and 70 % New York Heart Association Class II); 19 patients attended the second study visit and the median time between the two visits was 8 months. Pharmacokinetics were assessed using a nonlinear mixed-effects parent-metabolite model comprising two compartments for ramipril and one compartment for ramiprilat. The influence of body size and composition was best described by an allometric relationship with fat-free mass. In addition, ramipril clearance was related to patient age and daily ramipril dose, while clearance of ramiprilat was influenced by glome rular filtration rate and daily ramipril dose. There were no clinically relevant changes in the pharmacokinetics of ramipril and ramiprilat between the study visits. Due to the relatively stable pharmacokinetics of ramipril, regular outpatient visits at 6-month intervals seem appropriate to evaluate ramipril therapy.

The analysis of ramipril/ramiprilat concentration in human serum with liquid chromatography-tandem mass spectrometry - interpretation of high concentrations for the purposes of forensic toxicology.

Ramipril is a popular angiotensin-converting enzyme inhibitor applied in the treatment of hypertension. Its therapeutic effect is oriented on the concentration of the active metabolite ramiprilat. The information about toxic drug levels is missing in the literature. Therefore, the aim of this work was an indication of possible toxic ranges based on the analysis of real samples with high ramiprilat concentrations. For these purposes, an appropriate analytical LC-MS/MS method was developed and validated according to forensic guidelines and applied in the routine. Most real samples targeted for ramipril/ramiprilat were associated with the typical therapeutic drug range of 1-40 ng/mL described in the literature. However, higher drug levels with ramiprilat concentrations above 100 ng/mL could also be observed infrequently in cases of driving under the influence of drugs or attempted suicides. To the best of the author's knowledge, this is the first time antemortem ramipril and ramiprilat concentrations associated with driving under the influence of drugs and suicide attempts were discussed from a forensic point of view. The collected data enabled an indication of the ramiprilat toxic concentration range from about 600 ng/mL to at least 3500 ng/mL. The toxic concentration range discussed can be applied in the forensic practice as a reference for future cases.

Pharmacokinetics and pharmacodynamics of ramipril and ramiprilat after intravenous and oral doses of ramipril in healthy horses.

The pharmacokinetics and pharmacodynamics (PK/PD) of the angiotensin-converting enzyme inhibitor (ACEI) ramiprilat after intravenous (IV) and oral (PO) administration of ramipril have not been evaluated in horses. This study was designed to establish PK profiles for ramipril and ramiprilat as well as to determine the effects of ramiprilat on serum angiotensin converting enzyme (ACE) and to select the most appropriate ramipril dose that suppresses ACE activity. Six healthy horses in a cross-over design received IV ramipril 0.050 mg/kg, PO at a dose of 0 (placebo), and 0.050, 0.10, 0.20, 0.40 and 0.80 mg/kg ramipril. Blood pressures were measured and blood samples obtained at different times. Serum ramipril and ramiprilat concentrations and serum ACE activity were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectrophotometry, respectively. Systemic bioavailability of ramiprilat after PO ramipril was 6-9%. Percentages of maximum ACE inhibitions from baseline were 98.88 (IV ramipril), 5.31 (placebo) and 27.68, 39.27, 46.67, 76.13 and 84.27 (the five doses of PO ramipril). Blood pressure did not change during the experiments. Although oral availability of ramiprilat was low, ramipril has sufficient enteral absorption and bioconversion to ramiprilat to induce serum ACE inhibitions of almost 85% after a dose of 0.80 mg/kg ramipril. Additional research on ramipril administration in equine patients is indicated.

Ramipril protects the endothelium from high glucose-induced dysfunction through CaMKKß/AMPK and heme oxygenase-1 activation.

This study aims to investigate the effects of ramipril (RPL) on endothelial dysfunction associated with diabetes mellitus using cultured human aortic endothelial cells (HAECs) and a type 2 diabetic animal model. The effect of RPL on vasodilatory function in fat-fed, streptozotocin-treated rats was assessed. RPL treatment of 8 weeks alleviated insulin resistance and inhibited the decrease in endothelium-dependent vasodilation in diabetic rats. RPL treatment also reduced serum advanced glycation end products (AGE) concentration and rat aorta reactive oxygen species formation and increased aorta endothelium heme oxygenase-1 (HO-1) expression. Exposure of HAECs to high concentrations of glucose induced prolonged oxidative stress, apoptosis, and accumulation of AGEs. These effects were abolished by incubation of ramiprilat (RPT), the active metabolite of RPL. However, treatment of HAECs with STO-609, a CaMKKß (Ca(2+)/calmodulin-dependent protein kinase kinase-ß) inhibitor; compound C, an AMPK (AMP-activated protein kinase) inhibitor; and Zn(II)PPIX, a selective HO-1 inhibitor, blocked these beneficial effects of RPT. In addition, RPT increased nuclear factor erythroid 2-related factor-2 (Nrf-2) nuclear translocation and activation in a CaMKKß/AMPK pathway-dependent manner, leading to increased expression of the Nrf-2-regulated antioxidant enzyme, HO-1. The inhibition of CaMKKß or AMPK by pharmaceutical approach ablated RPT-induced HO-1 expression. Taken together, RPL ameliorates insulin resistance and endothelial dysfunction in diabetes via reducing oxidative stress. These effects are mediated by RPL activation of CaMKK-ß, which in turn activates the AMPK-Nrf-2-HO-1 pathway for enhanced endothelial function.

Liquid chromatography-tandem mass spectrometry method for simultaneous quantification of bisoprolol, ramiprilat, propranolol and midazolam in rat dried blood spots.

Dried blood spot (DBS) sampling represents a suitable method for pharmacokinetic studies in rats, particularly if serial sampling is needed. To study the pharmacokinetics of drugs in a rat heart failure (HF) model, we developed and validated a method for the simultaneous determination of bisoprolol, ramiprilat, propranolol and midazolam in DBS samples. Bisoprolol and ramipril are widely used in the treatment of HF, and midazolam and propranolol are markers of hepatic metabolism, which can be altered in HF. A 20µL sample of rat blood was pipetted onto Whatman 903 Protein Saver Card and allowed to dry. The whole spot was excised and 300µL of solvent (methanol with 10% ultrapure water and 0.1% formic acid) was added. After mixing and incubating the sample in an ultrasonic bath, a mixture of isotopically labeled internal standards was added. After centrifugation, the extracts were cleaned on an Ostro™ plate and analyzed using liquid chromatography-tandem mass spectroscopy. The method was successfully validated. No significant interference was observed in the retention times of analytes or internal standards. The intraday and interday accuracy and precision were within a ±15% interval. The method was linear in the range 5-250µg/L and the lower limit of quantification was 5µg/L for all four analytes. The absolute matrix effect ranged from 98.7% for midazolam to 121% for ramiprilat. The recovery was lowest for ramiprilat and highest for propranolol. Samples were stable at all tested temperatures. The method has been used successfully in a real-time pharmacokinetic study in rats.

Expressional profile of cardiac uncoupling protein-2 following myocardial ischemia reperfusion in losartan- and ramiprilat-treated rats.

BACKGROUND AND AIMS: The aim of this study was to investigate the early changes of cardiac uncoupling protein-2 (UCP2) expression following myocardial ischemia reperfusion in rats chronically treated with ramiprilat and losartan. METHODS: Male Wistar rats were assigned into seven groups (six in each): intact (control); sham-operated; nontreated rats subjected to ischemia and reperfusion (IR); ramiprilat-treated rats with (Ram+IR) and without ischemia (Ram); losartan treated with (Los+IR) and without ischemia (Los). Quantitative evaluation of UCP2 mRNA was carried out using real-time reverse transcription-polymerase chain reaction (RT-PCR). Mitochondria were isolated, and protein expression was quantified by Western blotting. RESULTS: In IR group: UCP2 protein but not mRNA level was increased in the ischemic area of the left ventricle (LV) (172% ± 26.7, p < 0.001 vs. LV of control). Following acute myocardial IR, UCP2 protein levels was increased in the ischemic area of the LV but not in RV, suggesting the local effect of ischemia on UCP2 expression. IR-induced overexpression of UCP2 was suppressed by ramiprilat and losartan. CONCLUSION: These findings suggest that losartan and ramiprilat can suppress UCP2 expression following myocardial IR, and by this mechanism may protect the myocardium against IR injury.

Perindopril and ramipril phosphonate analogues as a new class of angiotensin converting enzyme inhibitors.

A series of phosphonate analogues related to perindopril and ramipril were prepared and tested to estimate their ability to inhibit angiotensin converting enzyme. These new synthesized compounds were active ACE inhibitors with a promising activity.

High pulsatility flow stimulates smooth muscle cell hypertrophy and contractile protein expression.

Proximal arterial stiffening is an important predictor of events in systemic and pulmonary hypertension, partly through its contribution to downstream vascular abnormalities. However, much remains undetermined regarding the mechanisms involved in the vascular changes induced by arterial stiffening. We therefore addressed the hypothesis that high pulsatility flow, caused by proximal arterial stiffening, induces downstream pulmonary artery endothelial cell (EC) dysfunction that in turn leads to phenotypic change of smooth muscle cells (SMCs). To test the hypothesis, we employed a model pulmonary circulation in which upstream compliance regulates the pulsatility of flow waves imposed onto a downstream vascular mimetic coculture composed of pulmonary ECs and SMCs. The effects of high pulsatility flow on SMCs were determined both in the presence and absence of ECs. In the presence of ECs, high pulsatility flow increased SMC size and expression of the contractile proteins, smooth muscle α-actin (SMA) and smooth muscle myosin heavy chain (SM-MHC), without affecting proliferation. In the absence of ECs, high pulsatility flow decreased SMC expression of SMA and SM-MHC, without affecting SMC size or proliferation. To identify the molecular signals involved in the EC-mediated SMC responses, mRNA and/or protein expression of vasoconstrictors [angiotensin-converting enzyme (ACE) and endothelin (ET)-1], vasodilator (eNOS), and growth factor (TGF-ß1) in EC were examined. Results showed high pulsatility flow decreased eNOS and increased ACE, ET-1, and TGF-ß1 expression. ACE inhibition with ramiprilat, ET-1 receptor inhibition with bosentan, and treatment with the vasodilator bradykinin prevented flow-induced, EC-dependent SMC changes. In conclusion, high pulsatility flow stimulated SMC hypertrophy and contractile protein expression by altering EC production of vasoactive mediators and cytokines, supporting the idea of a coupling between proximal vascular stiffening, flow pulsatility, and downstream vascular function.

Tris(hydroxymethyl) aminomethane salt of ramipril: synthesis, structural characterization from X-ray powder diffraction and stability studies.

Tris(hydroxymethyl) aminomethane (tris) salt of API ramipril was synthesized, and characterized by FTIR, TG-DSC and ab initio X-ray powder structure analysis. The compound, ramipril-tris (II), crystallizes in the monoclinic space group P2(1) with a=24.3341(15), b=6.4645(5), c=9.5357(7) Å, ß=96.917(3)° and V=1489.1(3) Å(3). The crystal structure has been determined from laboratory X-ray powder diffraction data using direct space global optimization strategy (simulated annealing) followed by the Rietveld refinement. A network of intermolecular OH…O, CH…N and CH…O hydrogen bonds between the ramipril-ramipril, tris-tris and ramipril-tris components in the compound generates a two-dimensional molecular assembly in (110) plane. A comparative study of solid-state stabilities of ramipril-tris (II) with that of ramipril (I) and ramipril-erbumine (III) indicates that ramipril-tris (II) is the most stable one among the three, and the conversion to impurity D after 72 h at 80 °C is only 1.5%. The solution phase analysis at different pH values also reveals a greater stability of ramipril-tris (II) over ramipril (I).

Influence of ramiprilat and losartan on ischemia reperfusion injury in rat hearts.

UNLABELLED: HYPOTHESIS/INTRODUCTION: Our aim was to investigate whether a non-hypotensive dose of ramiprilat and losartan has myocardial protective effects during myocardial ischemia/reperfusion in vivo. MATERIALS AND METHODS: Three groups of rats were given 10 mg/kg per day of losartan for one (L-1W), four (L-4W) or 10 (L-10W) weeks. Another three groups were given 50 µg/kg per day of ramiprilat for one (R-1W), four (R-4W) or 10 (R-10W) weeks. The animals underwent 30 min of left anterior descending artery occlusion and subsequent reperfusion for 120 min. RESULTS: Myocardial infarct size (IS) was reduced in R-1W (28.4 ± 6.3%, p < 0.001), R-4W (27.8 ± 7.4, p < 0.001), L-4W (31.8 ± 6%, p < 0.05) and L-10W (25.3 ± 5.7, p < 0.001) groups compared with a saline group (48.3 ± 7.8%). A significant reduction in the number of ventricular ectopic beats (VEBs) was noted in groups R-1W (209 ± 41, p < 0.01), R-4W (176 ± 39, p < 0.01), L-4W (215 ± 52, p < 0.05) and L-10W (191 ± 61, p < 0.01 vs. saline 329 ± 48). The incidence of irreversible ventricular fibrillation (VF) and mortality were decreased significantly only in L-10W group. There were no significant decreases in episodes of VT, the incidence of irreversible VF and mortality in all of the groups treated with ramiprilat. CONCLUSION: These data indicate that losartan and ramiprilat protect the heart against ischemia/reperfusion injury independently of their hemodynamic effects but in a time-dependent manner.

Impact of methylation of acyl glucuronide metabolites on incurred sample reanalysis evaluation: ramiprilat case study.

BACKGROUND: Reanalysis of incurred samples showed that the bioanalytical method for the quantification of ramipril and ramiprilat was generating irreproducible results for ramiprilat. RESULTS: An additional peak interfering with ramiprilat was observed in the incurred samples but not in the calibrant and quality control samples. A similar interference was detected for ramipril, but it was chromatographically separated. Interferences were produced during sample preparation, which involves strong cation exchanger cartridges. The interfering products corresponded to the methylation of ramipril and ramiprilat glucuronide. CONCLUSION: Following this discovery, a reproducible method was developed and successfully validated for ramipril and ramiprilat. Additional stability tests were performed in the presence of glucuronide and diketopiperazine metabolites of ramipril and ramiprilat to demonstrate the method specificity.

Ramiprilate inhibits functional matrix metalloproteinase activity in Crohn's disease fistulas.

Increased expression of matrix metalloproteinase (MMP)-2, -3 and -9 has been demonstrated in Crohn's disease fistulas, but it is unknown whether these enzymes are biologically active and represent a therapeutic target. Therefore, we investigated the proteolytic activity of MMPs in fistula tissue and examined the effect of inhibitors, including clinically available drugs that beside their main action also suppress MMPs. Fistula specimens were obtained by surgical excision from 22 patients with Crohn's disease and from 10 patients with fistulas resulting from other causes. Colonic endoscopic biopsies from six controls were also included. Total functional MMP activity was measured by a high-pressure liquid chromatography (HPLC)-based, fluorogenic MMP-substrate cleavage assay, and the specific activity of MMP-2, -3 and -9 by the MMP Biotrak Activity Assay. The MMP inhibitors comprised ethylene-diamine-tetraacetic acid (EDTA), the synthetic broad-spectrum inhibitor, GM6001, the angiotensin-converting enzyme (ACE) inhibitor, ramiprilate, and the tetracycline, doxycycline. In Crohn's disease fistulas, about 50% of the total protease activity was attributable to MMP activity. The average total MMP activity was significantly higher (about 3.5-times) in Crohn's fistulas (471 FU/µg protein, range 49-2661) compared with non-Crohn's fistulas [134 FU/µg protein, range 0-495, (p < 0.05)] and normal colon [153 FU/µg protein, range 77-243, (p < 0.01)]. MMP-3 activity was increased in Crohn's fistulas (1.4 ng/ml, range 0-9.83) compared with non-Crohn's fistulas, [0.32 ng/ml, range 0-2.66, (p < 0.02)]. The same applied to MMP-9 activity [0.64 ng/ml, range 0-5.66 and 0.17 ng/ml, range 0-1.1, respectively (p < 0.04)]. Ramiprilate significantly decreased the average total MMP activity level by 42% and suppressed the specific MMP-3 activity by 72%, which is comparable to the effect of GM6001 (87%). Moreover, MMP-9 activity was completely blunted by ramiprilate. Doxycycline had no effect on MMP activity. Increased functional MMP activity, notably MMP-3 and -9, is present in Crohn's fistulas and may be inhibited by ramiprilate, a widely available ACE inhibitor.

Development and validation of UPLC tandem mass spectrometry assay for separation of a phase II metabolite of ramipril using actual study samples and its application to a bioequivalence study.

In this paper, we present a validated UPLC-MS/MS assay for determination of ramipril and ramiprilat from human plasma samples. The assay is capable of isolating phase II metabolites (acylglucornides) of ramipril from in vivo study samples which is otherwise not possible using conventional HPLC conditions. Both analytes were extracted from human plasma using solid-phase extraction technique. Chromatographic separation of analytes and their respective internal standards was carried out using an Acquity UPLC BEH C(18) (2.1 × 100 mm), 1.7 µm column followed by mass spectrometric detection using an Waters Quattro Premier XE. The method was validated over the range 0.35-70.0 ng/mL for ramipril and 1.0-40.0 ng/mL for ramiprilat.

Simultaneous determination of ramipril, ramiprilat and telmisartan in human plasma using liquid chromatography tandem mass spectrometry.

A rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed and validated for the simultaneous determination of ramipril, ramiprilat and telmisartan in human plasma. The solid-phase extraction technique was used for the extraction of ramipril, ramiprilat and telmisartan from human plasma. Trandolaprilat and hydrochlorothiazide were used as the internal standards (ISs). Chromatography was performed on a Hypurity C18, 5 µm, 50 mm × 4.6mm column, with the mobile phase consisting of ammonium acetate and acetonitrile (in a 20:80 ratio), followed by detection using mass spectrometry. The method involves a simple reversed isocratic chromatography condition and mass spectrometry detection, which enables detection at sub-nanogram levels. The method was validated and the lower limit of quantification for ramipril, ramiprilat and telmisartan was found to be 0.1 ng mL(-1), 0.1 ng mL(-1) and 2 ng mL(-1), respectively. The mean recovery for ramipril, ramiprilat and telmisartan ranged from 90.1 to 104.1%. This method increased the sensitivity and selectivity; resulting in high-throughput analysis of ramipril, ramiprilat and telmisartan using two different ISs in a single experiment for bioequivalence studies, with a chromatographic run time of 1.5 min only.

Effects of zofenopril and ramipril on cough reflex responses in anesthetized and awake rabbits.

Cough is the most common symptom reported by patients in a primary care setting and is one of the most frequent secondary effects recorded during treatment with angiotensin-converting enzyme (ACE) inhibitors. The aim of the current study was to analyze potential differences in cough induction between 2 structurally different ACE inhibitors, namely zofenopril, which has a sulphydryl moiety, and ramipril, which has a carboxyl moiety. The cough reflex was induced by chemical (citric acid) and/or mechanical stimulation of the tracheobronchial tree in awake and anesthetized rabbits. Intravenous injection of the active compounds of the 2 ACE inhibitors, zofenoprilat (288 nmol/kg) and ramiprilat (129 nmol/kg), caused similar hypotensive effects in anesthetized rabbits. None of the studied cough-related variables changed in response to ACE inhibitor administration, with the exception of the number of coughs. Ramiprilat, but not zofenoprilat, increased the cough response induced by both mechanical and chemical stimulation (1 mol/L citric acid aerosol) of the tracheobronchial tree. In awake animals, zofenoprilat- or vehicle-treated rabbits did not show any significant changes in the number of coughs induced by 1 mol/L citric acid aerosol compared to their respective basal values (from 15.2 ± 2.3 to 13.1 ± 1.3 and from 16.1 ± 4.9 to 15.8 ± 4.3, respectively). Conversely, ramiprilat resulted in a significant increase in the number of coughs (from 21.1 ± 2.6 to 34.9 ± 3.5; P < .01). These findings confirm that there are differences in the cough potentiation effect induced by different ACE inhibitors. The low rate of cough seen with zofenoprilat may be related to its ability to induce a lower accumulation of bradykinin and prostaglandins at the lung level.

Aliskiren versus ramipril in hypertension.

Aliskiren is an orally active direct renin inhibitor which inhibits the synthesis of angiotensin I by linking to active renin on a deep cleft of its molecular structure, the site of hydrolysis of the Leu10-Val11 bond of angiotensinogen. At variance with angiotensin-converting enzyme (ACE) inhibitors, aliskiren eliminates the main substrate for the 'escape' phenomenon (synthesis of angiotensin II from angiotensin I through alternative enzymatic pathways). The possibility that the antihypertensive effect of aliskiren differs from that of ACE inhibitors needs to be proved in specifically designed clinical trials. Over the past 2 years, three studies have been published which directly compared aliskiren with ramipril, in patients with hypertension. We made a pooled analysis of these studies. In order to avoid interference with additional drugs, analysis was restricted to trial periods when the two drugs were given as monotherapy. In each individual study, systolic blood pressure (BP) was slightly lower with aliskiren. Overall, systolic BP was lower with aliskiren than with ramipril (weighted mean difference between the treatments 1.84 mmHg; fixed effect model; p < 0.0001; and 1.87 mmHg; random effect model; p = 0.0055). The standardized mean difference between the treatments was 2.58 (fixed effect model; p < 0.0001) and 2.92 (random effect model; p = 0.0017) in favor of aliskiren. Compared with ramipril, aliskiren may have induced a more complete 'upstream' inhibition of the renin-angiotensin-aldosterone system, with consequent greater suppression of angiotensin II. Another potential explanation may be the longer terminal elimination halflife of aliskiren (about 40 hours) compared with ramiprilat (13-17 hours). These data provide further evidence that aliskiren monotherapy provides a sustained BP reduction over the 24 hours.

Sensitivity enhancement and matrix effect evaluation during summation of multiple transition pairs-case studies of clopidogrel and ramiprilat.

Sensitivity enhancement via summation of multiple MRM transition pairs is gaining popularity in tandem mass spectrometric assays. Numerous validation experiments describing the assays for two model substrates, clopidogrel and ramiprilat, were performed. The quantitation of clopidogrel was achieved by the summation of transition pairs m/z 322.2 to m/z 212.0 and m/z 322.2 to m/z 184.0, while that of ramiprilat was achieved by the summation of transition pairs m/z 389.2 to m/z 206.1 and m/z 389.2 to m/z156.1. The use of summation approach achieved sensitivities of >2 fold for both compounds as compared with the reported single MRM transition pair assays. The validation experiments addressed some important assay development issues, such as: (a) lack of impact of matrix effect; (b) unequivocal verification of the percentage contribution of each MRM transition pair towards sensitivity; (c) sensitivity enhancement factor achieved by summation approach of MRM transition pairs; and (d) accurate prediction of quality control samples using summation approach vs a single MRM transition pair. In summary, the appropriateness of using two MRM transition pairs for quantitation was demonstrated for both clopidogrel and ramiprilat. Additionally, pharmacokinetic application of the MRM transition pair assays using a summation approach was established for the two compounds.

Decreased renal perfusion rapidly increases plasma membrane Na-K-ATPase in rat cortex by an angiotensin II-dependent mechanism.

To understand how rapid changes in blood pressure can regulate Na-K-ATPase in the kidney cortex, we tested the hypothesis that a short-term (5 min) decrease in renal perfusion pressure will increase the amount of Na-K-ATPase in the plasma membranes by an angiotensin II-dependent mechanism. The abdominal aorta of anesthetized Sprague-Dawley rats was constricted with a ligature between the renal arteries, and pressure was monitored on either side during acute constriction. Left renal perfusion pressure was reduced to 70 +/- 1 mmHg (n = 6), whereas right renal perfusion pressure was 112 +/- 4 mmHg. In control (nonconstricted) rats (n = 5), pressure to both kidneys was similar at 119 +/- 6 mmHg. After 5 min of reduced perfusion, femoral venous samples were taken for plasma renin activity (PRA) and the kidneys excised. The cortex was dissected, minced, sieved, and biotinylated. Lower perfusion left kidneys showed a 41% increase (P < 0.003) in the amount of Na-K-ATPase in the plasma membrane compared with right kidneys. In controls, there was no difference in cell surface Na-K-ATPase between left and right kidneys (P = 0.47). PRA was 57% higher in experimental animals compared with controls. To test the role of angiotensin II in mediating the increase in Na-K-ATPase, we repeated the experiments (n = 6) in rats treated with ramiprilat. When angiotensin-converting enzyme was inhibited, the cell surface Na-K-ATPase of the two kidneys was equal (P =0.46). These results confirm our hypothesis: rapid changes in blood pressure regulate trafficking of Na-K-ATPase in the kidney cortex.

Bioanalytical method development and validation using incurred samples--simultaneous quantitation of ramipril and ramiprilat in human EDTA plasma by LC-MS/MS.

A new method development and validation approach is proposed in order to develop a reliable method for the simultaneous quantitation of ramipril and ramiprilat in the presence of numerous labile metabolites. This new approach involves the usage of a synthesized labile acyl glucuronide of ramipril as well as individual and pooled incurred (study) samples in the development and validation process. Following the method validation and prior to its application to a large clinical study, a mini pilot study was performed to evaluate the performance of the method. When the samples from the mini pilot study were analyzed by two different scientists, 100% of the results from incurred sample reanalysis (ISR) matched within 8% of difference and the mean differences were 0.21% and 1.40% for ramipril and ramiprilat, respectively. The validated concentration range reported in this article is 0.2-80 ng/mL for both analytes. Various stabilities, such as bench-top, autosampler, freeze/thaw, and long-term, were also successfully evaluated. The key to the success were low sample processing temperature (4 degrees C), proper choice of sample extraction procedure, and adequate chromatographic conditions to obtain good peak shape without the need of derivatization and baseline separation between the analytes and their glucuronide metabolites.

Pharmacokinetics and pharmacodynamics of ramipril and ramiprilat in healthy cats.

The pharmacokinetics of ramipril and its active metabolite, ramiprilat, was determined in cats following single and repeated oral doses of ramipril (Vasotop tablets) (once daily for 9 days) at dose rates of 0.125, 0.25, 0.5 and 1.0 mg/kg. The pharmacodynamic effects were assessed by measuring plasma angiotensin-converting enzyme (ACE) activity. Maximum ramipril concentrations were attained within 30 min following a single dose and declined rapidly (concentrations were below the limit of quantification 4 h after treatment). Peak ramiprilat concentrations were detected at approximately 1.5 h. The apparent terminal half-life (t((1/2)beta)) was > or =20 h irrespective of the dose. Ramiprilat accumulated in plasma (ratio of accumulation 1.3 to 1.9 depending on the dose rate) following repeated administration. Steady-state conditions were attained after the second dose. Excretion was predominant in faeces (87%) and to a lesser extent in urine (11%). The rate and extent of absorption of ramipril as well as its conversion to ramiprilat were not significantly influenced by the presence of food in the gastrointestinal tract. Plasma-ACE activity was almost completely abolished 0.5-2.0 h after treatment, irrespective of the dose rate. Significant inhibition of ACE activity of 54.7 to 82.6% (depending on the dosage) was still present 24 h after treatment. Treatment was well-tolerated in all cats. Ramipril at a dose rate of 0.125 mg/kg once daily produces significant and long-lasting inhibition of ACE activity in healthy cats. The appropriateness of this dosage regime needs to be confirmed in diseased cats.