Enhanced purification protocol for the angiotensin-converting enzyme from bovine systems and investigation of the in vitro effect of some active substances.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) synthesized by endothelial cells and responsible for the regulation of blood pressure was purified from the bovine lung with affinity chromatography method. The purification rate of the ACE of the bovine lung was calculated as 1748- fold. Optimum pH and optimum temperature for the purified ACE were found to be 7.6 and 35-40 °C, respectively. The purity and molecular weight of the ACE were designated with SDS-PAGE. The ACE was found to have three subunits with molecular weights of 57 kDa, 66 kDa, and 190 kDa. Then, the total molecular weight of the ACE was designated as 303 kDa with gel filtration chromatography. The effects of ACE inhibitors captopril, fosinopril, lisinopril, and beta-blockers propranolol, atenolol, and diuretic triamterene on ACE activity were studied. ACE inhibitors lisinopril, captopril, fosinopril, and diuretic triamterene demonstrated an inhibition effect on ACE activity. Beta-blockers indicated no effect on ACE. IC50 values of captopril, fosinopril, lisinopril, and triamterene from the graphical equation were calculated as 0.835 nM, 1.159 µM, 4.085 nM, and 227 µM, respectively. The inhibition type and Ki values of these compounds were determined from Lineweaver-Burk plots. Captopril, fosinopril, lisinopril, and triamterene demonstrated a non-competitive inhibition effect on ACE activity. Ki constants were found as 1.057 nM, 1.675 µM, 6.449 nM, and 419.5 µM, respectively. Captopril indicated the highest inhibitor effect with an IC50 value of 0.835 nM.

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.

Intermolecular interaction of fosinopril with bovine serum albumin (BSA): The multi-spectroscopic and computational investigation.

The intermolecular interaction of fosinopril, an angiotensin converting enzyme inhibitor with bovine serum albumin (BSA), has been investigated in physiological buffer (pH 7.4) by multi-spectroscopic methods and molecular docking technique. The results obtained from fluorescence and UV absorption spectroscopy revealed that the fluorescence quenching mechanism of BSA induced by fosinopril was mediated by the combined dynamic and static quenching, and the static quenching was dominant in this system. The binding constant, Kb , value was found to lie between 2.69 × 103 and 9.55 × 103  M-1 at experimental temperatures (293, 298, 303, and 308 K), implying the low or intermediate binding affinity between fosinopril and BSA. Competitive binding experiments with site markers (phenylbutazone and diazepam) suggested that fosinopril preferentially bound to the site I in sub-domain IIA on BSA, as evidenced by molecular docking analysis. The negative sign for enthalpy change (ΔH0 ) and entropy change (ΔS0 ) indicated that van der Waals force and hydrogen bonds played important roles in the fosinopril-BSA interaction, and 8-anilino-1-naphthalenesulfonate binding assay experiments offered evidence of the involvements of hydrophobic interactions. Moreover, spectroscopic results (synchronous fluorescence, 3-dimensional fluorescence, and Fourier transform infrared spectroscopy) indicated a slight conformational change in BSA upon fosinopril interaction.

Mitigation of experimental radiation nephropathy by renin-equivalent doses of angiotensin converting enzyme inhibitors.

PURPOSE: We tested five different angiotensin converting enzyme inhibitors (ACEI) as mitigators of experimental radiation nephropathy at drug doses calibrated to the plasma renin activity (PRA). This was done to determine whether all ACEI had the same efficacy as mitigators of radiation nephropathy when used at drug doses that gave equivalent suppression of the renin angiotensin system. METHOD: 10 Gy total body irradiation with bone marrow transplantation was used to cause radiation nephropathy in barrier-maintained rats. Equivalent ACEI doses were determined based on their effect to inhibit angiotensin converting enzyme (ACE) and raise the PRA in unirradiated animals. RESULTS: PRA-equivalent doses were found for captopril, lisinopril, enalapril, ramipril and fosinopril. These doses overlap the human doses of these drugs on a body surface area basis. All ACE inhibitors, except fosinopril, mitigated radiation nephropathy; captopril was a somewhat better mitigator than lisinopril, enalapril or ramipril. CONCLUSIONS: Most, but not all, ACEI mitigate radiation nephropathy at doses that overlap their clinically-used doses (on a body surface area basis). Fosinopril is known to be an ineffective mitigator of radiation pneumonitis, and it also does not mitigate radiation nephropathy. These pre-clinical data are critical in planning human studies of the mitigation of normal tissue radiation injury.

Characterization of stress degradation products of fosinopril by using LC-MS/TOF, MS(n) and on-line H/D exchange.

Fosinopril was subjected to stress degradation studies under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions. The drug underwent degradation in hydrolytic and photo acidic conditions, while it was stable to oxidative and thermal stress. The degradation products (DPs) were detected after using a gradient LC-MS method. These were characterized using a protocol involving comparison of mass fragmentation pathways of the drug and the DPs, and consideration of mass data generated during MS/TOF, MS(n) and H/D exchange studies. The degradation pathway of the drug was established, which was duly justified by mechanistic explanation.

Fosinopril-cyclodextrin inclusion complexes: phase solubility and physicochemical analysis.

Fosinopril is one of the most hydrophobic substances among the angiotensin-converting enzyme inhibitors, exhibiting low water solubility and poor bioavailability following oral administration. Inclusion complexes between the drug substance and cyclodextrins (CDs) were obtained in order to improve its solubility. The purpose of this study was to investigate the guest-host interaction of fosinopril sodium (FOS) with beta-cyclodextrin (beta-CD) and its derivative, randomly methylated beta-cyclodextrin (RAMEB) in solution by phase solubility diagrams (PSD) and in solid state by using thermal analysis, powder X-ray diffractometry (PXRD) and Fourier transform infrared spectroscopy (FTIR). The phase solubility analysis indicated that the solubility of FOS in simulated gastric fluid was increased in the presence of CDs and revealed for RAMEB an A(L)-type diagram, suggesting the formation of a 1:1 inclusion complex, and for beta-CD a B(s)-type phase diagram. The estimated apparent stability constant (K1:1), according to the Higuchi and Connors method, is 3209.99 M(-1) and 1770.34 M(-1) for RAMEB and beta-CD complexes respectively. The binary systems FOS/CDs were prepared using the kneading method in the molar ratio 1:1. The PXRD patterns and the thermograms indicated a drug amorphization process, higher for FOS/RAMEB binary system and the FTIR analysis suggested that the ester group of FOS is probably enclosed in the CD's cavity. The results of this study confirm the formation of inclusion complexes both in solution and in solid state and suggest that the complexes formation between FOS and CDs could improve the bioavailability of the drug due to the enhancing absorption expected from increased drug solubility.

Monitoring of fosinopril sodium impurities by liquid chromatography-mass spectrometry including the neural networks in method evaluation.

In this paper, the mass spectrometry (MS) detection has been applied for screening of fosinopril sodium impurities which arise during forced stress study. Before MS analysis, liquid chromatographic method with suitable mobile phase composition was developed. The separation was done on SunFire 100 mm x 4.6 mm 3.5 microm particle size column. The mobile phases which consisted of methanol-ammonium acetate buffer-acetic acid, in different ratios, were used in a preliminary study. Flow rate was 0.3 mL min(-1). Under these conditions, percent of methanol, concentration of ammonium acetate buffer and acetic acid content were tested simultaneously applying central composite design (CCD) and artificial neural network (ANN). The combinations of experimental design (ED) and ANN present powerful technique in method optimization. Input and output variables from CCD were used for network training, verification and testing. Multiple layer perceptron (MLP) with back propagation (BP) algorithm was chosen for network training. When the optimal neural topology was selected, network was trained by adjusting strength of connections between neurons in order to adapt the outputs of whole network to be closer to the desired outputs, or to minimize the sum of the squared errors. From the method optimization the following mobile phase composition was selected as appropriate: methanol-10 mM ammonium acetate buffer-acidic acid (80:19.5:0.5 v/v/v). This mobile phase was used as inlet for MS. According to molecular structure and literature data, electrospray positive ion mode was applied for analysis of fosinopril sodium and its impurities. The proposed method could be used for screening of fosinopril sodium impurities in bulk and pharmaceuticals, as well as for tracking the degradation under stress conditions.

Fosinopril H(2)-receptor antagonists interaction studies by derivative spectroscopy.

Fosinopril sodium, a phosphinic acid derivative is an angiotensin converting enzyme (ACE) inhibitor, which had been employed for the treatment of hypertension and congestive heart failure; long tem use of ACE inhibitor often result in stress ulcers due to which H(2) receptor antagonists are also concurrently prescribed. The later compete with histamine for H(2) receptors and block gastric acid secretion and some cardiovascular effects of histamine. Our studies are focused on the in vitro availability of fosinopril in presence of commonly used H(2) receptor antagonists. Derivative spectroscopy has been employed for the quantitation of fosinopril and H(2) receptor antagonists followed by linear regression analysis. These studies were carried out in buffers of pH 7.4 and 9 at 37, 48 and 60( masculine)C. Stability constant and thermodynamic function had also been calculated in order to evaluate the reaction mechanism. Commonly prescribed H(2) receptor antagonists like cimetidine, ranitidine and famotidine were used in these studies. Present study clearly indicated that most of the H(2) receptor antagonists studied decreased the availability of fosinopril which conclude that availability of fosinopril can be affected by the concurrent administration of H(2) receptor antagonists.

Método de cromatografía líquida de alta resolución para la determinación simultánea de fosinopril sódico e hidroclorotiazida en formulaciones de comprimidos / High performance liquid chromatographic method for simultaneous determination of fosinopril sodium and hydrochlorothiazide in tablets formulation

Se ha desarrollado un método HPLC de fase inversa con detección de UV sencillo, específi co, exacto y preciso para la determinación de fosinopril sódico e hidroclorotiazida en formulaciones farmacéuticas. La solución de fármaco se preparó en fase móvil y se inyectó en una columna C18 con detección de UV a 208 nm. La fase móvil era una mezcla de 0,2% p/v de ácido fosfórico en agua y acetonitrilo (30:70) añadida a una velocidad de fl ujo de 0,5 ml/min a 29 0C. Los gráfi cos de calibración fueron lineales en el rango de 10-50 μg/ml (r2 > 0,99) para el fosinopril sódico y de 6,25-31,35 μg/ml para la hidroclorotiazida (r2 > 0,99). El límite de detección fue de 0,5 μg/ml para el fosinopril sódico y de 1,0 μg/ml para la hidroclorotiazida, con un límite de cuantifi cación de 10 μg/ml y 6,25 μg/ml para el fosinopril sódico y la hidroclorotiazida, respectivamente. La evaluación estadística del método se examinó mediante calibración intradía (n=5) e interdía (n=5) y resultó satisfactoria, con una exactitud y precisión elevadas. El método sugerido se aplicó con éxito en la determinación simultánea de fosinopril sódico e hidroclorotiazida en formulaciones de comprimidos, con un elevado porcentaje de recuperación, buena exactitud y precisión

A simple, specifi c, accurate and precise reversed phase HPLC method with UV detection for the simultaneous determination of fosinopril sodium and hydrohclorthiazide in pharmaceutical formulations was developed. The drug solution was prepared in mobile phase and was injected into a C18 column with UV detection at 208 nm. The mobile phase was a mixture of 0.2%w/v phosphoric acid in water and acetonitrile (30:70) delivered at a fl ow rate of 0.5 ml/min at 29 0C. The calibration graphs were linear in the range of 10-50 μg/ml (r2 > 0.99) for fosinopril sodium and 6.25- 31.35 μg/ml for hydrochlorthiazide (r2 > 0.99). The detection limit was 0.5μg/ml for fosinopril sodium and 1.0 μg/ml for hydrochlorthiazide with quantitation limit of 10 μg/ml and 6.25 μg/ml for fosinopril sodium and hydrochlorthiazide, respectively. The statistical evaluation of the method was examined by performing intra-day (n=5) and inter-day calibration (n=5) and was found to be satisfactory, with high accuracy, and precision results. Application of the suggested method was successfully applied to the simultaneous determination of fosinopril sodium and hydrochlorthiazide in tablets formulation, with high percentage of recovery, good accuracy with precision

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.

Selection of solid dosage form composition through drug-excipient compatibility testing.

A drug-excipient compatibility screening model was developed by which potential stability problems due to interactions of drug substances with excipients in solid dosage forms can be predicted. The model involved storing drug-excipient blends with 20% added water in closed glass vials at 50 degrees C and analyzing them after 1 and 3 weeks for chemical and physical stability. The total weight of drug-excipient blend in a vial was usually kept at about 200 mg. The amount of drug substance in a blend was determined on the basis of the expected drug-to-excipient ratio in the final formulation. Potential roles of several key factors, such as the chemical nature of the excipient, drug-to-excipient ratio, moisture, microenvironmental pH of the drug-excipient mixture, temperature, and light, on dosage form stability could be identified by using the model. Certain physical changes, such as polymorphic conversion or change from crystalline to amorphous form, that could occur in drug-excipient mixtures were also studied. Selection of dosage form composition by using this model at the outset of a drug development program would lead to reduction of "surprise" problems during long-term stability testing of drug products.

Competitive inhibition of glycylsarcosine transport by enalapril in rabbit renal brush border membrane vesicles: interaction of ACE inhibitors with high-affinity H+/peptide symporter.

PURPOSE: To examine the inhibitory potential of enalapril [and other angiotensin converting enzyme (ACE) inhibitors] on glycylsarcosine (GlySar) transport by the high-affinity renal peptide transporter. METHODS: Studies were performed in rabbit renal brush border membrane vesicles in which the uptake of radiolabeled GlySar was examined in the absence and presence of captopril, enalapril, enalaprilat, fosinopril, lisinopril, quinapril, quinaprilat, ramipril and zofenopril. RESULTS: Kinetic analyses demonstrated that enalapril inhibited the uptake of GlySar in a competitive manner (Ki approximately 6 mM). Fosinopril and zofenopril had the greatest inhibitory potency (IC50 values of 55 and 81 microM, respectively) while the other ACE inhibitors exhibited low-affinity interactions with the renal peptide transporter. With respect to structure-function, ACE inhibitor affinity was strongly correlated with drug lipophilicity (r = 0.944, p < 0.001 for all ACE inhibitors; r = 0.983, p < 0.001 without enalaprilat, quinaprilat and quinapril). CONCLUSIONS: The data suggest that enalapril and GlySar compete for the same substrate-binding site on the high-affinity peptide transporter in kidney, and that ACE inhibitors can interact with the renal carrier and inhibit dipeptide transport.

The determination of palladium in fosinopril sodium (monopril) by ICP-MS.

A rapid, sensitive ICP-MS method was developed to determine palladium in fosinopril sodium. The assay could not be carried out in a purely aqueous solvent owing to the instability of the palladium species in this media. It was found that the most appropriate vehicle for solubilization of this material was a solution of 25% (v/v) 2-butoxyethanol and water. A minimum quantifiable limit of 0.1 microns g-1 for Pd in the sample (corresponding to 1 ng Pd mL-1 in the analyte solution) was obtained.

Aggregation behavior of fosinopril sodium--a new angiotensin-converting enzyme inhibitor.

Fosinopril sodium is an effective new angiotensin-converting enzyme (ACE) inhibitor that is very useful for the clinical treatment of hypertension. After oral administration, fosinopril sodium is only partially absorbed (about one third of the drug). We studied the solution behavior of fosinopril sodium in aqueous media by a combination of high-resolution nuclear magnetic resonance (NMR) spectroscopy and laser light scattering (LLS). LLS characterizes the self-association properties of fosinopril sodium in solution, and NMR chemical shifts provide information on molecular conformation and interactions. The results revealed that fosinopril sodium has a micelle-like self-association behavior with a critical micelle concentration (cmc) approximately 1.5 mg/mL. Hydrophobic interactions could induce formation of micellar aggregates, which had a narrow hydrodynamic size distribution, with an average diameter of approximately 150 nm at concentrations above the cmc. The surface activity and self-association of fosinopril sodium may be responsible for its early observed concentration-dependent stability in aqueous solution, unexpected decrease in solubility in the presence of metal ions, as well as the limited absorption in clinical studies.

A lamellar liquid crystal with fosinopril sodium.

The location and conformation of fosinopril sodium (FS) in a lamellar liquid crystal of water, sodium dodecyl sulfate, and decanol was studied by low-angle X-ray diffraction. The result showed the FS molecule to be located within the amphiphilic part of the liquid crystalline structure with the polar parts anchored at the water/polar part interface. An area per molecule in the range of 95-100 A2 showed not only the polar groups but also the benzene ring to be located at this interface.

Mechanism and kinetics of metal ion-mediated degradation of fosinopril sodium.

Fosinopril sodium (I), a new angiotensin converting enzyme inhibitor, is a diester prodrug of the active moiety II. We report here a novel transformation of fosinopril into beta-ketoamide, III, and a phosphonic acid, IV, mediated through metal ion participation. The interaction of fosinopril with magnesium ions was studied in a solution model system in which methanol was used as the solvent and magnesium acetate as the source of metal ions. Kinetic analysis indicated the degradation to be a bimolecular process, with the rate being first order in both metal ion and fosinopril concentration. The degradation products II, III, and IV effectively retarded the magnesium ion mediated reaction of fosinopril. Based on the results of 31P-NMR, 1H-NMR, Mn(II)-EPR spectroscopy experiments and mass spectrometry, a mechanism is postulated for this transformation. A key reactive intermediate has been characterized that supports the proposed mechanism. The results can account for the observed degradation profile of the fosinopril sodium in a prototype tablet formulation.