Sensitive SPE–HPLC method to determine a novel angiotensin-AT1 antagonist in biological samples
Introduction
The most of the peripheral effects of the renin–angiotensin system (RAS) is due to a biologically active component: Angiotensin II (AII). This is a major regulator of blood pressure, aldosterone secretion, and fluids homeostasis that is also an important etiological factor in hypertension and other cardiovascular disorders [1], [2]. In recent years, renin inhibitors with high specificity and affinity for human renin have been reported [3], but they have yet to be marketed.
An important class of anti hypertensive agents are represented by angiotensin converting enzyme (ACE) inhibitors that are effective for the treatment of most types of hypertension and congestive heart failure [4]. Nevertheless they possess some adverse effects, such as dry cough and angioedema [5].
A potentially advantageous approach to modulate the RAS is represented by a specific block of the AII actions at the receptor level [6]. The AT1 receptor subtype mediated virtually all of the known physiological actions of AII in cardiovascular, neuronal and other cells [7], [8], [9].
The first potent and orally active non-peptide AII antagonist was losartan (2-butyl-4-chloro-5-hydroxymethyl-1-[[2′-(1H-tetrazol-5-yl)biphenyl]-4-yl]methyl-1H-imidazole).
In order to develop new non-peptidic angiotensin AT1 antagonist, we recently synthesized CR3210 (C27H24N8) 4-[4-[(2-ethyl-5,7-dimethylimidazo[4,5-b]pyridin-3-yl)methyl]phenyl]-3-(2H-tetrazol-5-yl)quinoline [10]. This structure is closely related to losartan, and proved to act in vitro as an angiotensin II receptor antagonist. These substances have been developed in sequence to the angiotensin converting enzyme (ACE) inhibitors as a further therapeutic action on the renin–angiotensin–aldosterone system.
Reversed-phase high-performance liquid chromatography (HPLC) has been successfully used to determine benzylimidazole derivatives, i.e. sartane as losartan and its active metabolites in biological fluids [11], [12], [13], [14].
Our previous study reported the time profile of CR 3210 determined after intravenous and intraperitoneal administration to Sprague–Dawley rats [15]. The plasmatic time course indicated that the intraperitoneal administration of this drug was not useful to reach an adequate plasma concentration. Conversely, the intravenous administration gave an appreciable plasma concentration, even if the CR 3210 plasma level rapidly decreased after 15 min.
In consequence, the aim of this study was to define the pharmacokinetic profile of CR 3210 after oral administration, and to asses the urinary excretion over 36 h.
A high sensitive and selective analytical method was developed to determine this new drug in the nanogram range in plasma and urine samples.
Solid-phase extraction (SPE) was chosen to provide an efficient samples clean-up, that should not be underestimated, in particular for urine samples.
HPLC was used to determine CR 3210 and the internal standard (IS) CR 1505 (loxiglumide), 4-[(3,4-dichlorobenzoyl)amino]-5-[(3-methoxypropyl)pentylamino]-5-oxopentanoic acid, in plasma and urine of Sprague–Dawley rats. The method is simple and rapid and provides accurate and precise results.
Section snippets
Chemicals and standards
Acetonitrile, methanol and water HPLC grade (Carlo Erba, Milan, Italy) were used; deionized water was utilized to prepare buffers solution. All other reagents were of analytical-reagent grade (Carlo Erba, Milan, Italy).
CR 3210 was synthesized in our laboratories as previously described [8]; the internal standard CR 1505 was loxiglumide. Those compounds were supplied from Rotta Research Laboratorium (Monza, Italy). The structures of CR 3210 and CR 1505 are reported in Fig. 1.
Stock solutions (1
Results and discussion
The HPLC method proposed provides a simple procedure for the determination of a novel angiotensin-AT1 antagonist in biological samples.
The ultraviolet spectrum of CR 3210 shows two maxima (238 and 280 nm) and the internal standard CR 1505 has maximum at about 240 nm. A detection of 238 nm was chosen to detect both substances with a good sensitivity.
CR 3210 is a lipophilic molecule, where is present a quinoline bearing an acidic tetrazole moiety. In the structure of loxiglumide is present, as
Conclusions
The specific objective of this study was the determination of the new AT1-antagonist CR 3210 in rat plasma and urine.
The assay procedure based on a high performance liquid chromatography was fully validated. The method here described resulted sensitive and specific for the analysis of the test substance in biological samples.
The extraction procedures demonstrated a good efficiency. Solid-phase extraction allowed to isolate CR 3210 from rat plasma and urine with a good recovery. The accuracy
Acknowledgements
The authors are deeply grateful to Rotta Research Laboratorium (Monza, Italy) for the financial support. Furthermore the authors would thank Dr. N. Costa, supplier of plasma and urine samples for analyses.
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