Camptothecins in tumor homing via an RGD sequence mimetic.

A RGD peptide mimetic was conjugated to four camptothecins, with the purpose to improve their therapeutic index. The conjugate derivatives were evaluated against two tumor cell lines, one overexpressing integrins (human ovarian carcinoma, A2780) and a second one with a low integrin expression (human prostate cancer, PC3). The in vitro screening was completed with the adhesion behavior to vitronectin. Compound 8 (ST7456CL1) was selected for the in vivo investigation after stability tests over 24h, in PBS solution and in rat plasma, and compared to irinotecan. The former showed a prolonged half-life.

Efficient organic monoliths prepared by γ-radiation induced polymerization in the evaluation of histone deacetylase inhibitors by capillary(nano)-high performance liquid chromatography and ion trap mass spectrometry.

New monolithic HPLC columns were prepared by γ-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200 µm I.D.) and nano (100-75 µm I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8 min time windows, using fast gradients and very high linear flow velocities, up to 11 mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R_IC(50) and an averaged acetylation degree.

Extending the use of "Inverted Chirality Columns Approach" for enantiomeric excess determination in absence of reference samples: Application to a water-soluble camptothecin derivative.

The aim of the present study was to extend the use of the "Inverted Chirality Columns Approach (ICCA)" previously developed for the identification and quantitation of the trace enantiomer in highly enriched samples of the camptothecin (CPT) family of drugs to a novel water-soluble CPT derivative, namely namitecan (ST1968), currently undergoing phase I clinical trials as anticancer agent. Namitecan, identified from a series of hydrophilic 7-oxyiminomethyl derivatives, contains a free terminal amino group, which traditionally hampers the analysis under normal-phase HPLC conditions. Nevertheless, commercially available Pirkle-type chiral stationary phases (CSPs) available in both the enantiomeric forms (i.e., having the same bound selector with opposite configuration) mainly operate under normal-phase HPLC conditions. For this reason, namitecan was pre-column N-protected with isocyanates A-D and their sulfur analogues E-H to reduce its polarity by converting the amino group into a fragment compatible with the chiral recognition mechanism (i.e., ureido and thioureido groups). Once the optimal columns system and derivatizing agents were selected, an original enantioselective HPLC-MS/MS technique was developed on the Whelk-O1 CSPs.

Synthesis and characterization of novel internal surface reversed-phase silica supports for high-performance liquid chromatography.

An original synthetic method was developed for the preparation of a family of six novel deactivated restricted-access materials (RAMs), belonging to the group of the internal surface reversed-phase (ISRP) supports. The supports (ISRP-RAM phases A-F) have an alkyl-chain (14 methylenes) with two embedded ureido groups bound only to the internal surfaces of the porous silica, and polyvinyl alcoholic groups (PVA, 100,000-->22,000 molecular weight) chemically bound to the external surfaces. The average pore diameters of the prepared ISRP-RAM supports, calculated by inverse size-exclusion chromatography, ranged between 49 A and 88 A, and were able to exclude macromolecules heavier than about 24000 Da (such as serum proteins) from the pores. The novel supports were designed for the determination of a semi-synthetic anticancer drug of the camptothecin family in human plasma, but they represent universal ISRP-RAM supports not limited to such class of compounds.

Combination of HPLC "inverted chirality columns approach" and MS/MS detection for extreme enantiomeric excess determination even in absence of reference samples. Application to camptothecin derivatives.

An original, extremely sensitive and selective HPLC-MS/MS technique for the identification and determination of the minor enantiomer in nonracemic mixtures, even when only one enantiomer is available as reference, is described. The method is based on the so-called "inverted chirality columns approach" (ICCA) and consists of the use of chiral stationary phases (CSPs) available in both enantiomeric forms: in fact, inversion of the elution order for a pair of enantiomers is observed in response to the change in column chirality. This offers two key advantages: first, it is possible to demonstrate the potential enantioselectivity of the system by generating a virtual racemate, and second, it permits the choosing of the right column chirality for trace determination. Combination with MS/MS detection affords high specificity allowing not only high sensitivity (down to 0.0025% of the minor enantiomer) but also unequivocal peak identification in complex mixtures. Applications to semisynthetic derivatives of camptothecin, endowed with antitumor activity, are reported. Moreover, applicability of ICCA is not limited to this class of molecules but generates universal support. Its use might also be extended to other classes of compounds by using other CSPs, available in both enantiomeric forms.

Enantio- and chemo-selective HPLC separations by chiral-achiral tandem-columns approach: the combination of CHIROBIOTIC TAG and SCX columns for the analysis of propionyl carnitine and related impurities.

We describe a new tandem-columns chiral-achiral HPLC arrangement by using a chiral column (CHIROBIOTIC TAG) connected in series with an achiral column (Spherisorb S5 SCX), based on a strong cationic exchange mechanism; this approach is very useful for the analysis of chiral molecules, containing cationic groups in their structures. We used this special combination to develop an easy and convenient procedure for the enantio- and chemo-selective dosage of propionyl L-carnitine (1) and relative impurities (2-6), which allowed for the simultaneous separation and quantitation within 30 min. Under the best chromatographic conditions (acetonitrile-10 mM sodium dihydrogen phosphate 65:35, v/v (pHa 6.80) as the mobile phase and UV detection at 205 nm], all the individual peaks were well separated. The applicability of the method, fully validated, was demonstrated by the analysis of a pharmaceutical batch of propionyl L-carnitine, where we found the following contents: 98.5% for 1 (drug substance); 0.15% for 3; 0.1% for 5 and 0.2% for 6. The enantiomeric excess (e.e.%) measured for the drug substance was 98.9%. Finally, a single mixed-bed column, packed with a binary mixture of the chiral and achiral phases, in a 1:1 ratio, gave similar chromatographic results as the tandem-columns approach, and thus, offered an easy alternative solution to the separation of the considered mixture.

Enantioselective liquid chromatographic-electrospray mass spectrometric assay of beta-adrenergic blockers: application to a pharmacokinetic study of sotalol in human plasma.

An enantioselective high performance liquid chromatographic-electrospray ionization mass spectrometric (HPLC-ESI-MS) method for the direct determination of several beta-adrenergic blockers was developed and validated. The method is based on the direct separation of the enantiomers of drugs on a laboratory-made chiral stationary phase (CSP) containing covalently bonded teicoplanin (TE) as chiral selector. Detection of the effluent was performed by electrospray ionization mass spectrometry, run in the selected-ion recording (SIR) mode. The method was applied to the pharmacokinetic monitoring of sotalol (STL) in the plasma of five young healthy volunteers, dosed with racemic drug. The limits of quantitation (LOQ) reached 4 ng/ml for both sotalol enantiomers. Such a method, fully validated, offers a novel, fast and very efficient tool for the direct determination of sotalol enantiomers in human plasma, and can be generally applied to the beta-adrenergic blockers stereoselective pharmacokinetics.