A UPLC-DAD-Based Bio-Screening Assay for the Evaluation of the Angiotensin Converting Enzyme Inhibitory Potential of Plant Extracts and Compounds: Pyrroquinazoline Alkaloids from Adhatoda vasica as a Case Study

Angiotensin converting enzyme (ACE) plays a crucial role in regulating blood pressure in the human body. Identification of potential ACE inhibitors from medicinal plants supported the idea of repurposing these medicinal plants against hypertension. A method based on ultra-performance liquid chromatography (UPLC) coupled with a diode array detector (DAD) was used for the rapid screening of plant extracts and purified compounds to determine their ACE inhibitory activity. Hippuryl-histidiyl-leucine (HHL) was used as a substrate, which is converted into hippuric acid (HA) by the action of ACE. A calibration curve of the substrate HHL was developed with the linear regression 0.999. The limits of detection and quantification of this method were found to be 0.134 and 0.4061 mM, respectively. Different parameters of ACE inhibitory assay were optimized, including concentration, incubation time and temperature. The ACE inhibition potential of Adhatoda vasica (methanolic-aqueous extract) and its isolated pyrroquinazoline alkaloids, vasicinol (1), vasicine (2) and vasicinone (3) was evaluated. Compounds 1–3 were characterized by various spectroscopic techniques. The IC₅₀ values of vasicinol (1), vasicine (2) and vasicinone (3) were found to be 6.45, 2.60 and 13.49 mM, respectively. Molecular docking studies of compounds 1–3 were also performed. Among these compounds, vasicinol (1) binds as effectively as captopril, a standard drug of ACE inhibition.     

Improved method for direct high-performance liquid chromatography assay of angiotensin-converting enzyme-catalyzed reactions

A rapid and sensitive assay was developed for determination of the activity of angiotensin-converting enzyme (ACE) in the presence of inhibitory peptides present in soybean protein hydrolysates. The method utilizes reversed-phase high-performance liquid chromatography (HPLC) to separate and quantify hippuryl-histidyl-leucine (HHL) and hippuric acid (HA). HHL and HA were separated on a Symmetry C₁₈ column by gradient elution that used mixtures of trifluoroacetic acid (TFA)–acetonitrile and TFA–water as solvents. Analytical time and baseline separation of HA from HHL were improved over previous HPLC methods. In comparison to the standard spectrophotometric method, the new HPLC method obviates the need for ethyl acetate extraction of HA but requires direct injection of the ACE reaction mixture onto the HPLC column.     

Abnormal hepatobiliary clearance of 99mTc-N-(2,6-diethylphenylcarbamoylmethyl)iminodiacetic acid in the altered state of thyroid--by an analysis of deconvolution method

Hepatobiliary clearance of 99mTc-EHIDA was investigated in cases with altered thyroid function by deconvolution method. The results indicated that mean hepatic transit time of all control subjects revealed less than 10 minutes. On the other hand, mean hepatic transit time of cases with altered thyroid function revealed prolonged more than 13 minutes. Cases especially showing an elevated serum concentration of TSH compared with normal range (4.6 microU/ml) had a tendency of a high incidence of markedly prolonged mean hepatic transit time. These results suggest that thyroid hormone may influence on the hepatic metabolism of hepatobiliary radiopharmaceuticals. This phenomenon also could partly explain the cause of liver dysfunction seen in subjects with altered states.     

Prediction of glycyrrhizin disposition in rat and man by a physiologically based pharmacokinetic model

Three physiologically based pharmacokinetic models A--C, incorporating enterohepatic recycling, were developed to predict glycyrrhizin (GLZ) disposition in rat plasma and tissues, and human serum. Model A, which included fourteen compartments (artery, vein, tissues except brain, and gut lumen) with the assumption of direct excretion of GLZ from the liver into the gut lumen gave fairly good agreement between the observed and predicted disposition profiles in rat, but was unsuitable in man, where elimination is very rapid. Models B and C for man were obtained by adding a gallbladder compartment (drug storage organ) for the excretion from the liver into the gut lumen and by assuming continuous transfer from the storage compartment or instantaneous emptying from it during meal ingestion as the excretion process from the gallbladder into the gut lumen, respectively. The agreement between the observed and predicted serum concentration time-course profiles was better with model C than model B, especially in the terminal elimination phase, where secondary peaks appeared. However, it was thought that the observed serum disposition can be sufficiently well predicted by model B. In conclusion, prediction in rat was successful in all compartments except the brain, which shows a negligible distribution. Scale-up of the disposition kinetics of GLZ from rat to man was also successful.     

Absorption, first-pass metabolism, and disposition of itraconazole in rats

This study examined the pharmacokinetic disposition, oral absorption and hepatic extraction of itraconazole and its active metabolite, hydroxyitraconazole, in rats. After i.v. injection, serum itraconazole concentrations decreased biexponentially, with an average terminal elimination half-life, volume of distribution and systemic clearance of 4.9 h, 6.0 l/kg and 14.2 ml/min/kg, respectively. When given orally, its absorption was low, with a mean absolute bioavailability of 16.6%. The metabolite to parent drug area under the curve (AUC) ratio was higher after oral administration compared with i.v. injection (mean ratio, 2.7 vs. 0.9). The hepatic drug extraction ratio determined after femoral and portal vein administration averaged 18.5%. When hydroxyitraconazole was injected i.v., the elimination half-life, volume of distribution and systemic clearance of itraconazole averaged 10.0 h, 2.4 l/kg and 3.4 ml/min/kg, respectively. The fraction of the systemically available itraconazole that was metabolized to hydroxyitraconazole was 21.0% and 76.0% after i.v. and oral administration, respectively. In summary, this study is the first reporting the hepatic extraction of itraconazole and the i.v. disposition characteristics of hydroxyitraconazole in rats. Itraconazole is a drug with a low hepatic extraction ratio and its systemic clearance appears to be largely accounted for by hepatic metabolism.     

Synthesis, characterization and antioxidant activity of angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) catalyzes the conversion of angiotensin I (Ang I) to angiotensin II (Ang II). ACE also cleaves the terminal dipeptide of vasodilating hormone bradykinin (a nonapeptide) to inactivate this hormone. Therefore, inhibition of ACE is generally used as one of the methods for the treatment of hypertension. 'Oxidative stress' is another disease state caused by an imbalance in the production of oxidants and antioxidants. A number of studies suggest that hypertension and oxidative stress are interdependent. Therefore, ACE inhibitors having antioxidant property are considered beneficial for the treatment of hypertension. As selenium compounds are known to exhibit better antioxidant behavior than their sulfur analogues, we have synthesized a number of selenium analogues of captopril, an ACE inhibitor used as an antihypertensive drug. The selenium analogues of captopril not only inhibit ACE activity but also effectively scavenge peroxynitrite, a strong oxidant found in vivo.     

Analysis method of the angiotensin-I converting enzyme inhibitory activity based on micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC50) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity.     

Antioxidant activity of peptide-based angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) inhibitors are important for the treatment of hypertension as they can decrease the formation of vasopressor hormone angiotensin II (Ang II) and elevate the levels of vasodilating hormone bradykinin. It is observed that bradykinin contains a Ser-Pro-Phe motif near the site of hydrolysis. The selenium analogues of captopril represent a novel class of ACE inhibitors as they also exhibit significant antioxidant activity. In this study, several di- and tripeptides containing selenocysteine and cysteine residues at the N-terminal were synthesized. Hydrolysis of angiotensin I (Ang I) to Ang II by ACE was studied in the presence of these peptides. It is observed that the introduction of L-Phe to Sec-Pro and Cys-Pro peptides significantly increases the ACE inhibitory activity. On the other hand, the introduction of L-Val or L-Ala decreases the inhibitory potency of the parent compounds. The presence of an L-Pro moiety in captopril analogues appears to be important for ACE inhibition as the replacement of L-Pro by L-piperidine 2-carboxylic acid decreases the ACE inhibition. The synthetic peptides were also tested for their ability to scavenge peroxynitrite (PN) and to exhibit glutathione peroxidase (GPx)-like activity. All the selenium-containing peptides exhibited good PN-scavenging and GPx activities.     

A Pharmacokinetic Study of Mix-160 by LC-MS/MS: Oral Bioavailability of a Dosage Form of Citroflavonoids Mixture

This study was performed to evaluate and compare the pharmacokinetic parameters between two dosage formulations of hesperidin and naringenin: mixture and tablet. Our objective was to determine that the flavonoid tablet does not significantly modify the pharmacokinetic parameters compared with the mixture. For this study, we administered 161 mg/kg of either mixture (Mix-160) or tablet composed of hesperidin and by intragastric administration. Blood microsamples were collected from tail vein up to 24 h. Serum flavonoid extraction was performed by solid phase extraction and analyzed by LC-MS/MS of triple quadrupole (QqQ). Serum concentration vs. time plot showed that data fitted for a first-order model. The pharmacokinetic parameters were calculated by a noncompartmental model. The results showed that the absorption constant is higher than the elimination constant. The first concentration was found at five minutes, and minimal concentration at 24 h after administration, suggesting a enterohepatic recirculation phenomena and regulation of liver cytochromes’ activity. We did not find meaningful differences between the pharmacokinetic parameters of both samples. We concluded that tablet form did not interfere with the bioavailability of hesperidin and naringenin, and it could be a suitable candidate for developing a drug product.     

Pharmacokinetics of [6]-gingerol after intravenous administration in rats with acute renal or hepatic failure.

The pharmacokinetics of [6]-gingerol were investigated in rats with acute renal failure induced by bilateral nephrectomy, or those with acute hepatic failure induced by a single oral administration of carbon tetrachloride (CCl4), to clarify the contribution of the kidney and liver to the elimination process of [6]-gingerol. After bolus intravenous administration, a plasma concentration-time curve of [6]-gingerol was illustrated by a two-compartment open model. There was no significant difference in either the plasma concentration-time curve or any pharmacokinetic parameters between the control and nephrectomized rats. It is suggested, therefore, that renal excretion does not contribute at all to the disappearance of [6]-gingerol from plasma in rats. In contrast, hepatic intoxication with CCl4 elevated the plasma concentration of [6]-gingerol at the terminal phase. Its elimination half-life increased significantly, from 8.5 to 11.0 min, in CCl4-intoxicated rats. The extent of [6]-gingerol bound to serum protein was more than 90% and was affected very slightly by the CCl4-intoxication. These aspects indicate that [6]-gingerol is eliminated partly by the liver.     

Synthesis and biodistribution of bowman-birk soybean protease inhibitor conjugate with amphiphilic polyester

The modification of Bowman-Birk soybean protease inhibitor (BBI) with the monoaldehyde derivative of block copolymer of ethylene oxide and propylene oxide (PE),M _r 2000 is described. The conjugate contains five covalently bound polymer chains per protein molecule, and retains the ability to inhibit trypsin and chymotrypsinlike proteinases. The distribution of native BBI and the BBI-PE conjugate was examined in mice. After iv injection of [¹²⁵I]BBI and [¹²⁵I]BBIPE, both inhibitors distributed very rapidly to the liver, kidney, and lungs, and more slowly to the brain. At the same time-points (up to 24 h), radioactivity in the blood and organs of mice injected with modified inhibitor was higher than that of the native inhibitor. The blood concentration time profile following iv administration of two BBI preparations at a dose 3 mg/kg was reasonable well described by a two-compartment open model with first-order elimination kinetics. The total clearance of BBI-PE decreased by a factor of 8, body mean residence time increased by a factor of 5 in comparison with BBI. A physiological pharmacokinetic model was developed to describe the tissue-to-blood distribution of two inhibitors. One-compartment physiological organ model (flow limited) was used to describe of timecourse profiles of BBI concentration in organs. A two-compartment physiological organ model (membrane limited) was used to predict tissue-to-blood distribution of conjugated BBI in some organs of mice (liver, lungs). The predicted concentration curves of BBI and BBI-PE in blood and organs in mice (with the exception of kidney) showed good agreement with the observed values.     

Evaluation of postoperative hepatobiliary function by deconvolution analysis of hepatobiliary image data by 99mTc-N-pyridoxyl-5-methyltryptophan

Dynamic hepatobiliary image data were analyzed by the mathematical deconvolution to derive the transfer function (TF) which represents the impulse response function of the liver following direct bolus injection of a tracer into the liver. Biliary flow was evaluated by TF in patients with previous hepatobiliary surgery to detect abnormal flow causing problems such as cholangitis. Serial image data were collected for 60 min after intravenous injection of 37-75 MBq (1-2 mCi) of 99mTc-N-pyridoxyl-5-methyltryptophan (99mTc-PMT). TF was obtained by the matrix algorithm, using regional hepatogram as output and time-activity curve over the heart as input function. Minimum, mean and maximum transit time (Max. TT) were determined from TF. The functional image for each of the transit times was constructed by displaying the distribution of the values for matrix elements with a color scale. In this study, a critical Max. TT of 60 min was used to detect abnormal biliary flow. Of 30 cases, 9 positive cases were discriminated from 21 negative cases on the basis of the finding of presence or absence of areas with Max. TT of more than 60 min anywhere in the liver. Bowel activity overlapping the liver was excluded in the judgment. The validity of this method was evaluated in comparison with the clinical courses after surgery with (8 cases) or without (22 cases) problems. The results were sensitivity of 88%, specificity of 91%, accuracy of 90% and positive predictive value of 78%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oily drug carriers in cancer chemotherapy. II. Preparation of viscous ethyl oleate for intraarterial infusion therapy and its disposition in rats and hamsters.

Viscous ethyl oleate (VEO) was prepared as an oil drug carrier by the addition of aluminum stearate or ethyl cellulose. Since the rate of shear of VEO containing aluminum stearate was greatly and nonlinearly changed against the shearing stress compared to that containing ethyl cellulose, the latter was used for subsequent microvascular and organ distribution experiments in rats and hamsters. For infusion into the carotid artery in hamsters, neat ethyl oleate (EO, 4cP) or VEOs of various apparent viscosities (40, 80, 120 cP-VEOs) embolized the vascular system in the cheek pouch, although arrival time to the site where the embolization was observed and the embolization period differed depending on the type of oily drug carrier. For infusion into the hepatic artery in rats, however, only 120 cP-VEO embolized the vascular system in the liver. After infusion of the oily drug carrier containing 3H-oleic acid into the artery of hamster cheek pouch and rat liver, 30-50% of the radioactivity was gradually eliminated within 48 h, whereas about 80% of the dose was rapidly eliminated after infusion to rat stomach and kidney. In addition, the amount of 120 cP-VEO remaining in each organ 48 h after infusion was higher than those of EO and 40 and 80 cP-VEOs. Histological observation after infusion in rat liver revealed that 120 cP-VEO slowly migrated from the artery or arteriole to the sinusoidal capillary region. These results suggest that 120 cP-VEO can be used as a drug carrier because of its function of vascular embolization and high retention in a targeted tissue.     

Pharmacokinetics,hepatic disposition,and heart tissue distribution of 14 compounds in rat after oral administration of Qi-Li-Qiang-Xin capsule via ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry

In the present study, a specific and sensitive approach using ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry was developed and validated for the quantitative analysis of 14 constituents in rat plasma, liver, and heart. The method was fully validated and successfully applied to pharmacokinetic, hepatic disposition, and heart tissue distribution studies of 14 compounds after the oral administration of Qi-Li-Qiang-Xin capsule. Ginsenoside Rb1, alisol A, astragaloside IV, and periplocymarin were found to be highly exposed in rat plasma, while toxic components such as hypaconitine, mesaconitine, and periplocin had low circulation levels in vivo. Moreover, sinapine thiocyanate, neoline, formononetin, calycosin, and alisol A exhibited significant liver first-pass effects. Notably, high levels of alisol A, periplocymarin, benzoylmesaconine, and benzoylhypaconine were observed in the heart. Based on high exposure and appropriate pharmacokinetic features in the systemic plasma and heart, astragaloside IV, ginsenoside Rb1, periplocymarin, benzoylmesaconine, benzoylhypaconine, and alisol A can be considered as the main potentially effective components. Ultimately, the results provide relevant information for discovery of effective substances, as well as further anti-heart failure action mechanism investigations of Qi-Li-Qiang-Xin capsule.     

Acidity,lipophilicity, solubility,absorption, and polar surface area of some ACE inhibitors

Computational chemical methods have been used to correlate the molecular properties of the 10 ACE inhibitors (captopril, enalapril, perindopril, lisinopril, ramipril, trandolapril, quinapril, fosinopril, benazepril, and cilazapril) and some of their active metabolites (enalaprilat, perindoprilat, ramiprilat, trandolaprilat, quinaprilat, fosinoprilat, benazeprilat, and cilazaprilat). The computed pK _a values correlate well with the available experimental values. In the dicarboxylic ACE inhibitors, the carboxyalkyl carboxylate group of the ACE inhibitors studied is more acidic than the C-terminal carboxylate. However, at physiological pH = 7.4 both carboxyl groups of ACE inhibitors are completely ionized and the dicarboxyl-containing ACE inhibitors behave as strong acids. The available experimental partition coefficients of these ACE inhibitors investigated are well reproduced by the neural network-based ALOGPs and the fragment-based KoWWiN methods. All parent drugs (and prodrugs), with the exception of fosinopril, are compounds with low lipophilicity. Calculated pK _a, lipophilicity, solubility, absorption, and polar surface area of the most effective ACE inhibitors for the prevention of myocardial infarction, perindopril and ramipril, were found similar. Therefore, it is probable that the experimentally observed differences in the survival benefits in the first year after acute myocardial infarction in patients 65 years of age or older correlate closely to the physicochemical and pharmacokinetic characteristics of the specific ACE inhibitor that is used.     

Inhibition study of angiotensin converting enzyme by capillary electrophoresis after enzymatic reaction at capillary inlet

Capillary electrophoresis was used to study the inhibition of angiotensin-converting enzyme (ACE) by different inhibitors. Reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds. ACE activity was determined by the quantification of the reaction product, hippuric acid, at 230 nm. The technique was used to study the potency of five different inhibitors (captopril, lisinopril, perindoprilat, quinaprilat and benazeprilat). During a kinetic study, the Ki value of captopril was estimated to be 55.4 +/- 8.8 nM, a value consistent with previously reported values.     

On-line microdialysis coupled with microbore liquid chromatography with ultraviolet detection for continuous monitoring of free cefsulodin in rat blood

A microdialysis method followed by a microbore liquid chromatographic ultraviolet detection procedure has been performed for the assay of unbound cefsulodin in rat blood. A microdialysis probe was inserted into the jugular vein for blood sampling. This method involves an on-line design for submitting dialysate into the liquid chromatographic system. The chromatographic conditions consisted of a mobile phase of methanol-100 mM monosodium phosphoric acid (10:90, v/v, pH 5.0) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. Detection wavelength was set at 265 nm. Microdialysis probes, being laboratory-made, were screened for acceptable in vivo recovery while chromatographic resolution and detection were validated for response linearity as well as intra- and inter-day variabilities. The method was then applied to pharmacokinetics profiling of cefsulodin in the blood following intravenous administration of cefsulodin (20 mg/kg) in rats. Pharmacokinetics were calculated from the corrected data for dialysate concentrations of cefsulodin versus time. Based on pharmacokinetic calculation, cefsulodin best fitted to a two-exponential disposition. This study provided specific pharmacokinetic information for protein-unbound cefsulodin and demonstrated the applicability of this continuous sampling method for pharmacokinetic study.     

Validated ligand mapping of ACE active site

Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 A, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.     

LC‐MS/MS method for the determination of melamine in rat plasma: Toxicokinetic study in Sprague–Dawley rats

Most recently, melamine has raised international concern for its catastrophic health effects stemming from tainted infant formula. So far there is limited information concerning the pharmacokinetics of melamine in mammals. The present report concerns the development and validation of a sensitive HPLC‐ESI‐MS/MS method for the pharmacokinetic study of melamine in rat. The method employed a simple liquid–liquid extraction process for plasma sample cleanup, and the extraction recoveries of melamine from plasma were consistent at different concentrations. There was a linear relationship between chromatographic area and concentration over the range of 10–5000 ng/mL for melamine in plasma (R = 0.995). In this work, for the first time, melamine was administered intravenously and orally to Sprague–Dawley rats and the pharmacokinetic characteristics of this contaminant were investigated. The mean values of major pharmacokinetic parameters of oral availability, the mean steady‐state distribution volume (V_ss), clearance, and plasma elimination half‐life (T_1/2) of melamine in Sprague–Dawley rats were 72.9 ± 13.2%, 102.5 ± 12.5 mL/kg, 20.1 ± 3.8 mL/h/kg, and 4.9 ± 0.5 h, respectively. The rats pharmacokinetic study results suggested that melamine was predominantly restricted to blood or extracellular fluid and is not extensively distributed to most organ tissues. Meanwhile, melamine should be primarily eliminated by renal filtration for rats and does not undergo significant metabolism. These data should be useful to regulatory for risk assessment.     

Gas chromatographic-mass spectrometric assessment of the pharmacokinetics of amineptine and its main metabolite in volunteers with liver impairment

A pharmacokinetic study of amineptine (Survector) and its C5 metabolite, resulting from a beta-oxidation of the heptanoic acid side-chain, was undertaken with ten human volunteers, who received a single 100-mg tablet of amineptine orally. They were affected with liver impairment in order to determine if this situation would alter greatly the pharmacokinetic parameters. The internal standard was the octanoic acid homologue. Analyses were carried out by gas chromatography (GC) and GC-mass spectrometry using TMS ester derivatives. Plasma samples were extracted using a C18 reversed-phase cartridge at pH 4.0. Mass fragmentographic measurements on the plasma samples were performed on the m/z ions (M + H)+ and (base peak)+ using ammonia chemical ionization. The global evaluation of precision was good and the coherence between the two modes of measurements, (base peak)+ and (M + H)+ ions, gave a regression factor r close to unity. For amineptine the total body clearance and mean residence time were accurate and precise with eight volunteers, but only four volunteers showed such coherent data for the slope of the elimination curve, beta, and half-life. However, the beta value, half-life and mean residence time of the C5 metabolite were accurate and precise with seven, eight and ten volunteers, respectively. It is concluded that the drug was still detoxified at normal levels.