Gabriele Caccialanza

Isolation and characterization of bioactive compounds from plant resources: The role of analysis in the ethnopharmacological approach

The phytochemical research based on ethnopharmacology is considered an effective approach in the discovery of novel chemicals entities with potential as drug leads. Plants/plant extracts/decoctions, used by folklore traditions for treating several diseases, represent a source of chemical entities but no information are available on their nature. Starting from this viewpoint, the aim of this review is to address natural-products chemists to the choice of the best methodologies, which include the combination of extraction/sample preparation tools and analytical techniques, for isolating and characterizing bioactive secondary metabolites from plants, as potential lead compounds in the drug discovery process. The work is distributed according to the different steps involved in the ethnopharmacological approach (extraction, sample preparation, biological screening, etc.), discussing the analytical techniques employed for the isolation and identification of compound/s responsible for the biological activity claimed in the traditional use (separation, spectroscopic, hyphenated techniques, etc.). Particular emphasis will be on herbal medicines applications and developments achieved from 2010 up to date.

Validation of a RP-LC method for the simultaneous determination of isoniazid, pyrazinamide and rifampicin in a pharmaceutical formulation.

A simple and accurate liquid chromatographic method was developed and validated for estimation of isoniazid (ISN), pyrazinamide (PYR) and rifampicin (RIF) in combined dosage forms. Drugs were chromatographed on a reverse phase C18 column using a mobile phase gradient and monitored at the corresponding maximum of each compounds. Peaks were identified with retention time as compared with standards and confirmed with characteristic spectra using diode-array detector. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method does not require any specific sample preparation except the use of a guard column. The method is linear (r(2)>0.999), precise (RSD%: 0.50% for ISN, 0.12% for PYR and 0.98% for RIF), accurate (overall average recovery yields: 98.55% for ISN, 98.51 for PYR and 98.56% for RIF) and selective. Due to its simplicity and accuracy the method is suitable for routine quality control analysis of antitubercolosis combination dosage form.

Capillary electrophoresis investigation of a partially unfolded conformation of β2-microglobulin

Dialysis‐related amyloidosis is a disease in which partial unfolding of β2‐microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of β2‐microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full‐length β2‐microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full‐length β2‐microglobulin.

Pharmaceutical strategies against amyloidosis: Old and new drugs in targeting a protein misfolding disease

The group of diseases caused by abnormalities of the process of protein folding and unfolding is rapidly growing and includes diseases caused by loss of function as well as diseases caused by gain of function of misfolded proteins. Amyloidoses are caused by gain of function of certain proteins that lose their native structure and self-assemble into toxic insoluble, extracellular fibrils. This process requires the contribution of multiple factors of which only a few are established, namely the conformational modification of the amyloidogenic protein, proteins post-translational modifications and the co-deposition of glycosaminoglicans and of serum amyloid P component. In parallel with the exponential growth of biochemical data regarding the key events of the fibrillogenic process, several reports have shown that small molecules, through the interaction with either the amyloidogenic proteins or with the common constituents, can modify the kinetics of formation of amyloid fibrils or can facilitate amyloid reabsorption. These small molecules can be classified on the basis of their protein target and mechanism of action, according to the following properties. 1) molecules that stabilize the amyloidogenic protein precursor 2) molecules that prevent fibrillogenesis by acting on partially folded intermediates of the folding process as well as on low molecular weight oligomers populating the initial phase of fibril formation 3) molecules that interact with mature amyloid fibrils and weaken their structural stability 4) molecules that displace fundamental co-factors of the amyloid deposits like glycosaminoglycans and serum amyloid P component and favor the dissolution of the fibrillar aggregate.

CE can identify small molecules that selectively target soluble oligomers of amyloid β protein and display antifibrillogenic activity

Soluble and toxic oligomers of amyloid β (Aβ) protein have been identified as the true neurotoxic species involved in Alzheimers disease and considering them as targets to inhibit Aβ aggregation might have a therapeutic value. We previously set up a CE method that enables the separation and quantification of transient oligomers of Aβ protein‐containing 42 amino acids (Aβ1–42) along the pathway leading to fibrils and we now demonstrate how this method can be successfully applied to examine the in vitro inhibitory effects of small molecules on Aβ oligomerization. To this end, we investigated mitoxantrone and pixantrone, two well‐known anticancer drugs, as well as suramin and a suramin‐like compound. By using CE, it is here shown how mitoxantrone and pixantrone either reduce or block Aβ1–42 oligomerization, while Thioflavin T spectrofluorimetric assay and transmission electron microscopy demonstrate how these two compounds also display antifibrillogenic activity. Interestingly, in vitro cell viability experiments indicated that pixantrone significantly reduces Aβ1–42 neurotoxicity.

Egg yolk riboflavin binding protein as a new chiral stationary phase in high-performance liquid chromatography

A chiral stationary phase for high-performance liquid chromatography based on hen egg yolk riboflavin binding protein is introduced. The purified protein was immobilized on activated 5NH2 Nucleosil silica. Chiral acidic, basic and uncharged drugs were chromatographed and the influence of the mobile phase parameters on the retention times and enantioselectivity was studied. Thirteen out of the twenty compounds tested were partially or baseline resolved. These encouraging preliminary results suggest that this chiral stationary phase may be applicable to a wide range of drug enantiomers in the reversed-phase mode.

Target-Based Drug Discovery: the Emerging Success of Frontal Affinity Chromatography Coupled to Mass Spectrometry

Frontal affinity chromatography coupled to mass spectrometry (FAC–MS) has been reported as a potential method for screening compound mixtures against immobilized target proteins. The potentiality of this analytical approach is described and illustrated with a number of examples based on targets of pharmaceutical interest.

Evaluation of a penicillin G acylase-based chiral stationary phase towards a series of 2-aryloxyalkanoic acids, isosteric analogs and 2-arylpropionic acids

The chiral recognition properties of a new chiral stationary phase based on immobilized penicillin G acylase were investigated using 35 acidic racemates. Twenty-seven compounds were resolved with high separation factors. The influences of mobile phase pH, type of organic modifier and ionic strength on enantioselective retention were studied. The most important tool for affecting the enantioselectivity was the mobile phase pH and interestingly the retention order of the enantiomers of some analytes could be controlled by this parameter. The analysis time for resolving enantiomers could be adjusted with a minor decrease in enantioselectivity using a high ionic strength mobile phase buffer while both retention and enantioselectivity decreased by adding organic modifier to the mobile phase. Displacement studies have demonstrated that the enzymatically active site and the chiral adsorption site overlap.

Evaluation of stereoselective dissolution of verapamil hydrochloride from matrix tablets press-coated with chiral excipients

Abstract In most cases the modulation of the drug delivery rate from modified-release formulations is achieved with polymers also used as chiral stationary phases in liquid chromatography. It is therefore hypothesized that the interaction of the enantiomers with the excipient may lead to differentiated delivery rates from the devices for each enantiomer. This study evaluates the stereoselective dissolution of (±)-verapamil, a model racemic drug and, for this purpose, different matrix compositions, a commercial product and a particular delivery device have been considered. The delivery device, recently proposed for the delayed release of drugs, consists of an active core containing the drug, coated by compression with different types of chiral polymeric materials. The quantitative determination of verapamil enantiomers released by these systems was carried out using a stereospecific HPLC method. Hydroxypropylmethylcellulose, β-cyclodextrin, hydroxypropyl-β-cyclodextrin and cross-linked amylose did not show any stereoselective dissolution properties while pectin, galactomannan and scleroglucan seemed to give a slightly higher dissolution rate of the R, compared with the S enantiomer. It is, however, to be verified whether these small differences in the release rate of the two enantiomers detected ‘in vitro’ could lead to real ‘in vivo’ effects.

Evaluation of quail egg white riboflavin binding protein as a chiral selector in high-performance liquid chromatography and capillary electrophoresis

A new chiral stationary phase for high-performance liquid chromatography of quail egg white riboflavin binding protein is presented. Several chiral acidic, basic and uncharged drugs were analysed and the influence of the mobile phases parameters on the retention times and enantioselectivity was evaluated. On the basis of the results obtained, the same protein was studied as a background electrolyte additive in free solution capillary electrophoresis, in order to evaluate if capillary electrophoresis (CE) could be used as a rapid scouting technique for screening the enantioselectivity of novel proteins without immobilisation on a solid support. To investigate if it is possible to directly compare the results obtained by each technique, the CE experiments were planned on the basis of both the findings and ideas originated in liquid chromatography.