Suitability of DPPH spiking for antioxidant screening in natural products: the example of galloyl derivatives from red maple bark extract.

To investigate the antioxidant potential in natural products, radical scavenging tests (ABTS, DPPH, ORAC, etc.) are usually considered as the first approach. In addition to the standard colorimetric assays, methods using separation techniques (on-line and pre-column assays) have been developed in the past decades. Based on the peak area (PA) reductions of compounds monitored by HPLC, the pre-column spiking method allows rapid characterisation of natural matrices avoiding laborious isolation steps. However, available information about the significance of the results produced remains scarce. Here, we report, for the first time, a discussion of the potential of the pre-column DPPH spiking method to pinpoint antioxidant compounds using red maple bark extract (RMBE). First, DPPH spiking was conventionally applied to the galloyl compounds in the extract showing the inadequacy of assessing results by PA reductions. The method was then applied to pure galloyl derivatives, evaluating their molar amount reacted (MAR) for more significance. The comparison with the standard DPPH-HPLC/AE method directly monitoring DPPH• inhibition highlighted the inability to retrieve the respective antioxidant efficiencies (AE) of each compound by using DPPH spiking. Despite its limitations, the DPPH spiking method brought to light an autoxidation phenomenon and a matrix/mixture effect investigated through tertiary mixtures of galloyl compounds. Although restricted to the compounds from one natural matrix, this study questions the validity of the spiking method as usually performed and could serve as a basis for further investigations (explorations of other natural products, kinetics considerations). Graphical abstract Investigation of the pre-column DPPH spiking method through the case of galloyl derivatives.

Structural model for phenylalkylamine binding to L-type calcium channels.

Phenylalkylamines (PAAs), a major class of L-type calcium channel (LTCC) blockers, have two aromatic rings connected by a flexible chain with a nitrile substituent. Structural aspects of ligand-channel interactions remain unclear. We have built a KvAP-based model of LTCC and used Monte Carlo energy minimizations to dock devapamil, verapamil, gallopamil, and other PAAs. The PAA-LTCC models have the following common features: (i) the meta-methoxy group in ring A, which is proximal to the nitrile group, accepts an H-bond from a PAA-sensing Tyr_IIIS6; (ii) the meta-methoxy group in ring B accepts an H-bond from a PAA-sensing Tyr_IVS6; (iii) the ammonium group is stabilized at the focus of P-helices; and (iv) the nitrile group binds to a Ca(2+) ion coordinated by the selectivity filter glutamates in repeats III and IV. The latter feature can explain Ca(2+) potentiation of PAA action and the presence of an electronegative atom at a similar position of potent PAA analogs. Tyr substitution of a Thr in IIIS5 is known to enhance action of devapamil and verapamil. Our models predict that the para-methoxy group in ring A of devapamil and verapamil accepts an H-bond from this engineered Tyr. The model explains structure-activity relationships of PAAs, effects of LTCC mutations on PAA potency, data on PAA access to LTCC, and Ca(2+) potentiation of PAA action. Common and class-specific aspects of action of PAAs, dihydropyridines, and benzothiazepines are discussed in view of the repeat interface concept.

Pharmacophoric features and Ca2+ ion holding capacity of verapamil.

Ab initio Hartree-Fock calculations have been performed at the 6-31G level to study the pharmacophoric features of verapamil. Both the unprotonated and the protonated forms of verapamil have been studied. The study predicts that the drug enters the body in protonated form and is anchored to the receptor via H-bond formation involving protonated amine. Huge conformational change as well as deprotonation is required before the drug is capable of holding Ca(2+) ions. Folded form of drug is capable of holding Ca(2+) ion and the chiral center also seems to be involved to certain extent.

Pharmacokinetics and pharmacodynamics of R- and S-gallopamil during multiple dosing.

AIMS: Using a stable isotope technique we investigated the pharmacokinetics and pharmacodynamics of gallopamil after administration of 50 mg pseudoracemic gallopamil every 12 h for 7 doses (72 h). METHODS: Six male healthy volunteers were studied. After the seventh dose the pharmacokinetics and pharmacodynamics were assessed. Serum levels of gallopamil were measured by gas chromatography/mass spectrometry. Effects of gallopamil were measured by ECG recording. RESULTS: The apparent oral clearances (R: 4.8 l min-1 (95% CI: 2.9-6.8); S: 5.5 l min-1 (95% CI: 2.5-8.5)) and half-lives (R: 6.2 h; S: 7.2 h) of R- and S-gallopamil were similar (P >0.05). The serum protein binding (fu R: 0.035 (95% CI: 0.026-0. 045); S: 0.051 (95% CI: 0.033-0.069)) and the renal elimination (% of dose R: 0.49%; S: 0.71%) were enantioselective. Gallopamil had a potent effect on the PR interval (% prolongation 35.7% (95% CI: 14. 0-57.3)). No changes in other electrocardiographic or cardiovascular parameters were observed. CONCLUSIONS: The pharmacokinetics and bioavailability of the racemic drug gallopamil are not stereoselective at steady-state and are therefore not substantially altered compared with the single dose administration of gallopamil.

Identification of human cytochrome P-450 isoforms involved in metabolism of R(+)- and S(-)-gallopamil: utility of in vitro disappearance rate.

Isoforms of cytochrome P-450 (CYP) involved in the metabolism of gallopamil enantiomers were identified by measuring the disappearance rate of parent drug from an incubation mixture with human liver microsomes and recombinant human CYPs. Mean (+/- S.D.) intrinsic clearances (CL(int)) of R(+)- and S(-)-gallopamil in human liver microsomes were 0.320 +/- 0.165 and 0.205 +/- 0.107 ml/min/mg protein, respectively. These values were highly correlated with the 6beta-hydroxylation activity of testosterone, a marker substrate of CYP3A4 (r = 0.977 and 0.900 for R(+)- and S(-)-gallopamil, respectively, p <.001). Ketoconazole and troleandomycin, selective inhibitors of CYP3A4, and polyclonal antibodies raised against CYP3A4/5 markedly reduced the CL(int) of gallopamil enantiomers in human liver microsomes. Among the 10 recombinant human CYP isoforms, CYP3A4 exhibited the highest CL(int) of gallopamil enantiomers, and CYP2C8 and CYP2D6 also exhibited appreciable activity. When the contribution of CYP3A4 to the total metabolic clearance of gallopamil enantiomers in human liver microsomes was estimated by relative activity factor, the mean (+/- S.D.) contributions were 92 +/- 18 and 68 +/- 19% for R(+)- and S(-)-gallopamil, respectively. These values were comparable to the rates of immunoinhibition by antibodies raised against CYP3A4/5 observed in human liver microsomes. The present study suggests that CYP3A4 is a major isoform involved in the overall metabolic clearance of gallopamil enantiomers in the human liver, and that the present approach based on disappearance rate may be applicable to identify major isoforms of CYP involved in the metabolism of a drug in human liver microsomes.

Properties of the racemic species of verapamil hydrochloride and gallopamil hydrochloride.

It is well known that the stereoselective actions associated with the enantiomeric constituents of a racemic drug can differ markedly in their pharmacodynamic or pharmacokinetic properties. Nevertheless, molecular chirality manifests itself in the solid, that is, crystalline state. The aim of this work was to characterize the solid-state properties of verapamil HCl and gallopamil HCl, two well-known chiral calcium channel antagonists. The characterization of the solid state for the single enantiomers and equimolecular mixtures for both the calcium antagonists was performed by solid-state techniques such as Fourier transform infrared (FT-IR spectroscopy), X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC). The FT-IR spectra and XRD of the single enantiomers are different from those of the corresponding equimolecular mixture owing to their different crystalline structure. The thermal behavior of the racemates and pure enantiomers were examined by DSC, and the resultant experimental and theoretical binary phase diagrams are discussed. Spectroscopic solid-state techniques, such as FT-IR and XRD, are useful in combination with thermal analysis for characterizing the racemic species of chiral drugs. The data obtained prove that the equimolecular mixtures of both verapmil hydrochloride and gallopamil hydrochloride exist as racemic compounds. Determination of the enantiomeric purity of the enantiomers and racemic compounds of both the calcium antagonists analyzed was performed by DSC.

Conformational analysis of free and Ca(2+)-bound forms of verapamil and methoxyverapamil.

In a recent experimental study (Tetreault, S. and Ananthanarayanan, V.S. (1993) J. Med. Chem. 36, 1324-1332) we showed that verapamil can bind Ca2+ in a nonpolar medium to form 1:1 and 2:1 drug:Ca2+ complexes and proposed that such complexes may represent the bioactive form of the drug. A similar suggestion has also been made earlier from theoretical considerations of the geometry of the drug (Zhorov, B. and Govyrin, V. (1983), Dokl.Akad.Nauk SSSR 273, 497-501). In order to fully understand the nature of the drug-Ca2+ complex, we present in this paper a systematic conformational analysis of the protonated and neutral forms of verapamil and one of its potent analogues, methoxyverapamil (D600). For each form of verapamil and D600, the energies and generalized coordinates of all minimum-energy conformations (MECs) with the energy less than 5 kcal/mol above the global minimum have been accumulated and sorted in the order of increasing energies. A protocol was then used to search in the files MECs meeting a set of geometrical criteria and to sum up their populations. The geometrical criteria involved the predisposition of the oxygen and nitrogen atoms of the drug molecule to form bi- tri- and tetradentate complexes with Ca2+. Use of these criteria demonstrated that both verapamil and D600 have several low-energy structural patterns that are predisposed for bi- and polydentate chelation of Ca2+. Models of various types of 1:1 drug:Ca2+ complexes as well as two models of 2:1 drug:Ca2+ "sandwich" complex were obtained. Such models may be biologically relevant in understanding the nature of the ternary complex formed by the drug, Ca2+ and the calcium channel.

Comparison of lowest energy conformations of dimethylcurine and methoxyverapamil: evidence of ternary association of calcium channel, Ca2+, and calcium entry blockers.

Verapamil and dimethylcurine are Ca2+ entry blockers of essentially different chemical structures which presumably bind to the same arylalkylamine receptor of the L-type Ca channel. A systematic conformational analysis of methoxyverapamil (D-600) and dimethylcurine has been carried out using a molecular mechanics method. The lowest minimum-energy conformations of D-600 are predisposed to chelate Ca2+ by four oxygen atoms of the stacked methoxyphenyl moieties. Comparison of the lowest energy conformations of D-600-Ca2+ and dimethylcurine revealed a similar spatial disposition of cationic groups and methoxyphenyl moieties in the two compounds. A three-dimensional model of arylalkylamine receptor was suggested which incorporates two nucleophilic areas of the Ca channel. Dimethylcurine binds to these areas by its quaternary amine functions, whereas D-600 does so by amine function and via coordinated Ca2+. The results support the hypotheses on ternary complex formation between the ligands of Ca channel, their receptors, and Ca2+.

Enantioselective gallopamil protein binding.

The protein binding of the enantiomers of gallopamil has been investigated in solutions of human serum albumin, alpha 1-acid glycoprotein and serum. Over the range of concentrations attained after oral gallopamil administration, the binding of both enantiomers to albumin, alpha 1-acid glycoprotein, and serum proteins was independent of gallopamil concentration. The binding to both human serum albumin (40 g/liter) [range of fraction bound (fb) R: 0.624 to 0.699; S: 0.502 to 0.605] and alpha 1-acid glycoprotein (0.5 g/liter) (range of fb R: 0.530 to 0.718; S: 0.502 to 0.620) was stereoselective, favoring the (R)-enantiomer (predialysis gallopamil concentrations 2.5 to 10,000 ng/ml). When the enantiomers (predialysis gallopamil concentration 10 ng/ml) were studied separately in drug-free serum samples from six healthy volunteers the fraction of (S)-gallopamil bound (fb: 0.943 +/- 0.016) was lower (P < 0.05) than that of (R)-gallopamil (fb: 0.960 +/- 0.010). The serum protein binding of both (R)- and (S)-gallopamil was unaffected by their optical antipodes (fb R: 0.963 +/- 0.011; S: 0.948 +/- 0.015) indicating that at therapeutic concentrations a protein binding enantiomer-enantiomer interaction does not occur. The protein binding of (R)- and (S)-gallopamil ex vivo 2 h after single dose oral administration of 50 mg pseudoracemic gallopamil (fb R: 0.960 +/- 0.010: predialysis [R] 6.9 to 35.3 ng/ml; S: 0.943 +/- 0.016: predialysis [S] 9.5 to 30.7 ng/ml) was comparable to that observed in vitro in drug-free serum. Gallopamil metabolites formed during first-pass following oral administration, therefore, do not influence the protein binding of (R)- or (S)-gallopamil.

Application of a variance-stabilizing transformation approach to linear regression of calibration lines.

A variance-stabilizing transformation (VST) was applied to the linear regression of calibration standards of different drugs in plasma. This transformation involved the normalization of the dependent variable peak height or peak area ratio (Y), and the independent variable, plasma drug concentration (C). This transformation led to a constant variance in the regression error term across the measured concentration range and allowed the evaluation of the unbiased slope and y intercept with minimum variance. The utility of the VST procedure in comparison with the ordinary least squares (OLS) approach, routinely used in pharmaceutical studies for constructing calibration lines, is described. The principal advantage of the VST approach is allowing a lower minimum level of drug quantification while using a single calibration line over a wide range of drug concentrations. The VST method is especially useful to quantify drug plasma levels in pharmacokinetic evaluation of sustained-release dosage forms, where the precise quantification of low levels of drug is critical. The application of the VST method was explored and evaluated in comparison with the OLS method for pharmacokinetic assays of diltiazem, gallopamil, nitroglycerin, and nicotine.