Synergistic interaction of novel lactate dehydrogenase inhibitors with gemcitabine against pancreatic cancer cells in hypoxia.

BACKGROUND: Hypoxia is a driving force in pancreatic-ductal-adenocarcinoma (PDAC) growth, metastasis and chemoresistance. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis, ensuring supply of energy and anabolites in hypoxic-environments. Therefore, we investigated the molecular mechanisms underlying the pharmacological interaction of novel LDH-A inhibitors in combination with gemcitabine in PDAC cells. METHODS: Lactate dehydrogenase A levels were studied by quantitative RT-PCR, western blot, immunofluorescence and activity assays in 14 PDAC cells, including primary-cell-cultures and spheroids, in normoxic and hypoxic conditions. Cell proliferation, migration and key determinants of drug activity were evaluated by sulforhodamine-B-assay, wound-healing assay, PCR and LC-MS/MS. RESULTS: Lactate dehydrogenase A was significantly increased under hypoxic conditions (1% O2), where the novel LDH-A inhibitors proved to be particularly effective (e.g., with IC50 values of 0.9 vs 16.3 µM for NHI-1 in LPC006 in hypoxia vs normoxia, respectively). These compounds induced apoptosis, affected invasiveness and spheroid-growth, reducing expression of metalloproteinases and cancer-stem-like-cells markers (CD133+). Their synergistic interaction with gemcitabine, with combination index values <0.4 in hypoxia, might also be attributed to modulation of gemcitabine metabolism, overcoming the reduced synthesis of phosphorylated metabolites. CONCLUSION: Lactate dehydrogenase A is a viable target in PDAC, and novel LDH-A inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours.

MET as a potential target for the treatment of upper gastrointestinal cancers: characterization of novel c-Met inhibitors from bench to bedside.

The receptor tyrosine kinase mesenchymal-epithelial transition factor (c-Met) plays a pivotal role in regulation of cell proliferation and migration. Abnormal expression of c-Met has been associated with poor prognosis in several cancer types, including upper gastrointestinal malignancies. Moreover, c-Met interaction with multiple signalling pathways involved in tumor growth and invasive/metastatic phenotype has gained substantial attention in the last few years, suggesting the therapeutic potential of this target. This has led to the development and evaluation of a number of c-Met inhibitors. Here we describe the critical role of the HGF/c-Met pathway in cancer, as well as the preclinical and clinical investigations on c-Met inhibitors in solid tumors, with particular emphasis on recent findings with small-molecule inhibitors in gastrointestinal cancers. Clinical trials with several of these novel inhibitors have been encouraging and one of them, crizotinib (dual c-Met/ALK inhibitor), has recently been approved for lung cancers with ALK-rearrangement. There are accumulating evidences on the therapeutic potential of this and other c-Met inhibitors for the treatment of other malignancies, such as gastric and pancreatic cancers. These inhibitors might be used in combination with chemotherapy as well as with other biological agents, in order to overcome different resistance mechanisms. However, further studies are needed to identify determinants of the activity of c-Met inhibitors, through the analysis of genetic and environmental alterations affecting c-Met and parallel pro-cancer pathways. These studies will be critical to improve the efficacy and selectivity of current and future anticancer strategies targeting c-Met.

Definition and system implementation of strategies for method development of chiral separations in normal- or reversed-phase liquid chromatography using polysaccharide-based stationary phases.

This paper proposes strategies in normal- and reversed-phase liquid chromatography (NP-HPLC or NPLC and RP-HPLC or RPLC), which were developed using three polysaccharide-based stationary phases. Those strategies are implemented in a knowledge-based system for the chiral separation of drug enantiomers. Each strategy includes a screening and an optimisation stage. The screening stage allows a fast evaluation of separation possibilities and enantioselectivity for many drugs in a short period of time, while the optimisation stage gives the opportunity to enhance, if needed, the initially obtained separation. Different examples demonstrate the effectiveness of the strategies for fast method development.

Comparison of the miniaturised techniques capillary electrochromatography and capillary liquid chromatography for the chiral separation of chlorthalidone.

The aim of this study was to compare the miniaturised techniques, capillary electrochromatography (CEC) and capillary liquid chromatography (CLC), for the chiral separation of chlorthalidone. In both cases, hydroxypropyl-beta-cyclodextrin was used as a chiral selector in the mobile phase, while an achiral stationary phase was used. Earlier, this separation was already optimised in CEC. Now, the separation was optimised in CLC. The influence of the organic modifier content and the cyclodextrin concentration on the separation was studied by means of a central composite design. Optimal separation conditions were determined, after response modelling, from the response surface contour plots. When these conditions were compared with those of the CEC optimisation, we can see the potential of using CLC as a chiral separation technique since less chiral selector was used, faster separations were obtained and better repeatability was observed in comparison with its electrical-driven counterpart.

Screening approach for chiral separation of pharmaceuticals part II. Reversed-phase liquid chromatography.

A screening strategy for the rapid separation of drug enantiomers by reversed-phase liquid chromatography was developed using three cellulose/amylose stationary phases. The key point to achieve enantioselectivity is the control of the compound ionisation. Only two mobile phases, i.e. an acidic phosphate buffer (pH 2.0) containing a chaotropic salt (KPF6) and a borate buffer (pH 9.0) mixed with acetonitrile, are used in the proposed strategy. This strategy was successfully applied to a set of 37 diverse chiral pharmaceuticals. Satisfactory enantioselectivity was achieved for 89% of them.

Screening approach for chiral separation of pharmaceuticals. Part I. Normal-phase liquid chromatography.

A strategy for rapid screening for the separation of chiral molecules of pharmaceutical interest by normal-phase liquid chromatography using three cellulose/amylose stationary phases is proposed. In a first step, the most important parameters for the separations were determined and studied for their effects by means of experimental designs. Results showed that the cellulose tris-(3,5-dimethylphenylcarbamate), the amylose tris-(3,5-dimethylphenylcarbamate) and the cellulose tris-(4-methylbenzoate) stationary phases have very broad and complementary enantiorecognition properties. The type of organic modifier used in the mobile phase appeared to have a dramatic influence on the quality of the separation. Based on the results of the preliminary study, a screening strategy was developed and successfully applied to a set of 36 diverse drugs. Enantiomeric separation was observed in 89% of cases and the analysis times were usually shorter than 20 min.

Knowledge-based system for method development of chiral separations with capillary electrophoresis using highly-sulphated cyclodextrins.

Method development for chiral separations is not easy because it requires experience and many experimental possibilities can be chosen. In order to help the analyst, a knowledge-based system (KBS) for the rapid determination of experimental parameters, which allow a baseline separation of enantiomers, has been developed. On the basis of own laboratory knowledge, completed with literature data, rules were defined and a KBS was built. Five different techniques are considered in this KBS. This paper describes the capillary electrophoresis (CE) section, in which a strategy has been defined based on the use of highly-sulfated cyclodextrins as chiral selectors. A structured representation of the knowledge and its implementation in Toolbook software is presented.

Rapid screening for chiral separations by short-end injection capillary electrophoresis using highly sulfated cyclodextrins as chiral selectors.

Two series of amino acid derivatives and phenylamines were used to evaluate the potential of highly sulfated cyclodextrins (HS-CDs) for the screening for chiral separations by capillary electrophoresis (CE). HS-CDs showed to be very versatile and to exhibit very high enantioselectivity. The use of short-end injection allowed to reduce dramatically the analysis time. From the results obtained, a scheme for the rapid screening of enantiomeric molecules was developed and applied to various chiral drugs. Results are very satisfying as almost all compounds (62 out of 67) could be baseline-resolved. Usually, less than three experiments were necessary to obtain very good separation.

Fast development of separation methods for the chiral analysis of amino acid derivatives using capillary electrophoresis and experimental designs.

The use of experimental design in method development was studied for the chiral separation of several amino acid derivatives with capillary electrophoresis. The aim of this study was to define rapidly experimental conditions under which the enantiomers can be sufficiently separated for quantification and to derive a methodology for the separation of new compounds. Three modified cyclodextrins (CDs) were used as chiral selectors: hydroxypropyl-beta-cyclodextrin, carboxymethyl-beta-CD and sulfobutylether-beta-CD. The following factors were examined: the type of cyclodextrin, the CD concentration, the pH and the % of organic modifier (methanol) of the electrolyte. Two types of fractional factorial design were used depending on the type of analyte and on the number of factors selected: a 3(4-2) fractional factorial design (4 factors studied at 3 different levels) and a 2(3-1) fractional factorial design (3 factors at 2 different levels). From the 14 compounds investigated, 12 could be separated with one or another CD and not more than 9 experiments were required. No generalisation of the best analysis conditions was possible within this family of compounds. Specific analysis conditions must be defined for each analyte. Experimental designs have shown to be very useful to determine rapidly conditions under which each enantiomer can be separated with an acceptable resolution.

Rapid development of the enantiomeric separation of beta-blockers by capillary electrophoresis using an experimental design approach.

A rapid method for determining the separation conditions for chiral resolution of eleven beta-blocking drug substances by capillary electrophoresis is described, using an experimental design approach. An acidic phosphate-triethanolamine buffer and an uncoated fused-silica capillary were used for all experiments. Several modified cyclodextrins were applied as chiral selectors: sulfobutyl ether beta-cyclodextrin (SBE-beta CD), dimethyl beta-cyclodextrin (DM-beta CD), carboxymethyl beta-cyclodextrin (CM-beta CD), and hydroxypropyl beta-cyclodextrin (HP-beta CD). Two different fractional factorial experimental designs were applied: (1) a design examining four factors at three levels (3(4-2)) and (2) one examining three factors at two levels (2(3-1)). The factors studied were: type of cyclodextrin, cyclodextrin concentration, pH of the background electrolyte and percentage of organic modifier. Enough resolution for the separation of the enantiomers and even for their quantification was reached. The same scheme is proposed when a fast chiral separation method needs to be developed for other drug families.

Purification and characterization of a novel protease from culture filtrates of a Streptomyces sp.

A fibrinolytic protease has been isolated from Streptomyces sp. culture filtrate by successive chromatography on Mono S and Sephadex G50. The purified protease had a molecular mass of 33 kDa and had an isoelectric point of 6.7. It showed a sharp pH optimum at 7.8 with maximal protease activity between 35 degrees C and 50 degrees C. Its amino acid composition and amino-terminal sequence (17 residues) were determined. The protein exhibited marked hydrolytic activity toward the substrates N-Succ-(Ala)2-Pro-Phe-pNA (K(m) = 0.77 mM, Vmax = 24.2 mumol mg-1 min-1) and N-Succ-(Ala)2-Pro-Leu-pNA (K(m) = 0.92 mM, Vmax = 7.7 mumol mg-1 min-1). It was totally inhibited by alpha 1-antitrypsin, D-Phe-Pro-Arg-chloromethylketone and sodium dodecyl sulfate but was insensitive to EDTA, dithiothreitol, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, pepstatin or elastatinal. In this respect, this protease differed in its physico-chemical and biochemical properties from other extracellular proteases previously found in bacteria and fungi. The results suggest that it has properties of chymotrypsin-like serine-type proteases.

Two-site immunoassay of recombinant hirudin based on two monoclonal antibodies.

Two monoclonal antibodies (mAbs), 10-2 and 10-5, both directed against recombinant hirudin variant 2-Lys47 (rHV2), were selected for their high affinity and epitopic specificities to develop a two-site immunoassay of rHV2. The mAb concentrations, incubation time, and temperature were optimized. The immunoassay has a detection limit for rHV2 of 45 ng/L in plasma and 30 ng/L in urine. The reactivity of the mAbs was tested against rHV2 and several forms of this protein truncated in the carboxyl terminus. The capture mAb 10-2 was found to be mainly directed against rHV2, whereas tracer mAb 10-5 was independent of the carboxyl-terminal region of the protein. This explains the high specificity of the immunoassay for the 65-amino acid form of hirudin.

Characterization of the deamidated forms of recombinant hirudin.

Recombinant hirudin variant rHV2-Lys 47 (MW = 6906.5) was intentionally deamidated by incubation in pH 9 phosphate buffer at 37 degrees C. Anion-exchange HPLC analysis showed that 11 forms could be generated. These were isolated and purified by combined anion-exchange and reversed-phase HPLC. Acid-catalyzed carboxyl methylation was used to introduce a mass shift of +15 amu per deamidated residue present in the molecule before analysis by liquid secondary ion mass spectrometry (LSIMS). Methylation enhanced, in particular, the abundance of the sequence ions in the LSIMS spectra. This permitted the determination of both the number (three) and the localization of the deamidated residues: Asn 52, Asn 53, and a residue located in the N-terminal 1-39 domain. Complementary sequencing techniques proved that the latter residue was Asn 33. Altogether four mono-, three di-, and four tri-deamidated forms were identified. The heterogeneity of the forms having identical deamidation positions but being chromatographically separable is thought to arise from the generation of alpha- and beta-aspartyl iso forms during the nonenzymatic deamidation process.

Liquid secondary ion mass spectrometry applied to structural confirmation of enzymically prepared C-terminal-truncated derivatives of recombinant hirudin.

The thrombin-specific inhibitor, hirudin variant rHV2-Lys 47 (rHirudin), is a 65-amino acid polypeptide produced by recombinant DNA technology in yeast. Previous studies have shown that the acidic C-terminal segment of hirudin is susceptible to enzymic degradation. To address the question of C-terminal-truncated forms of the protein in terms of by-products or metabolites, well-defined reference compounds are needed. We prepared nine derivatives by carboxypeptidase Y digestion of rHirudin followed by a two-step chromatographic purification. Liquid secondary ion mass spectrometric measurements performed on peptides collected after reversed-phase high-performance liquid chromatography showed three pure forms (1-64, 1-63 and 1-56) and three mixtures of two forms each (1-62 + 1-61, 1-58 + 1-57 and 1-55 + 1-54), which were readily distinguished from one another by their mass spectra. Further purification of these co-eluted samples was achieved by ion-exchange chromatography and their structures were confirmed by liquid secondary ion mass spectrometry. Preliminary studies conducted on intact rHirudin indicated that this is an excellent analytical tool for mass measurements of hirudin-related proteins. Indeed, it allowed rapid (within 10-15 min), precise (0.50 a.m.u. relative to expected value), reproducible (mean MH+ = 6907.64 +/- 0.42 a.m.u.), sensitive (up to 500 ng, i.e. 72 pmol) and specific measurement of the quasi-molecular ion (MH+) of the protein, and was thus readily applicable to the analysis of several derivatives.

Site specific radioiodination of recombinant hirudin.

Recombinant hirudin variant rHV2-Lys47 was radioiodinated using the chloramine-T method. Depending on the reaction pH, the two tyrosine residues, Tyr3 and Tyr63, responded differently to iodination but without change in total iodination yield. Of the incorporated -125 iodine 80% was located on Tyr3 at pH 7.4, but 65% was found on Tyr63 at pH 4. These distinct iodination patterns suggest the existence of a pH-dependent multimerization and/or important conformational changes in the tertiary structure with pH. Each radiotracer was purified to high specific activity by simple low-pressure chromatography including gel filtration and reverse-phase separation, both on short cartridges. The method was validated by reverse-phase and anion-exchange HPLC with on-line radioactivity detection. The iodination sites were characterized following carboxypeptidase Y cleavage coupled with radio-HPLC.

Pharmacokinetic study of 14C-radiolabelled elliptinium acetate in patients with metastatic breast cancer.

The pharmacokinetics of 14C-labelled elliptinium acetate (Celiptium) was studied in 3 patients with metastatic breast cancer after a 1-h intravenous infusion of the drug at the dose of 100 mg/m2. Total radioactivity measurements were done in plasma, urine and faeces over 120 h. Plasma peak concentration of radioactivity reached during the infusion was 1.6 +/- 0.2 micrograms eq/ml. A comparison with previously published results showed a high ratio of plasma total radioactivity to unchanged drug levels. This could indicate a fast metabolic transformation of the drug. Radioactivity profile in plasma reincreased 8 h after the infusion, strongly suggesting a contribution of biliary radiolabelled metabolites to an enterohepatic cycle. This phenomenon was not observed on the plasma curve of unchanged drug levels, thus indicating that the drug itself and its known conjugates (glucuronide and cysteinyl derivatives) contributed only to a minor extent to the enterohepatic circulation of the drug. More than 70% of radioactivity was recovered in urine and faeces during 120 h for two of the three patients, and fecal excretion appeared to be the major route of elimination of the drug in humans.

Rapid enzymatic degradation of [125I] (Tyr 10) FGF (1-10) by serum in vitro and involvement in the determination of circulating FGF by RIA.

In the conditions used in the RIA procedure for circulating FGF quantitation, the tracer [125I] (Tyr 10) FGF (1-10) was extensively degraded into two non immunoreactive peptides corresponding to a sequential removal of two amino acid residues at the NH2-terminus i.e. Pro and Ala. A FGF like immunoreactive fraction exists in serum the molecular weight of which was estimated to be 240 Kda. This fraction was also able to perform the same extensive degradation of (Tyr 10) FGF (1-10) than whole serum. The results presented raise the question of the validity of RIA for the determination of circulating FGF. They also present evidence that a high molecular weight serum fraction which reacts as immunoreactive FGF is an enzymatic activity responsible for biodegradation of the growth factor rather than a distinct biological entity which is related to the FGF structure.

Oxidative biotransformation of the antitumour agent elliptinium acetate: Structural characterization of its human and rat urinary metabolites.

The electrophilic properties of the antitumour drug N(2)-methyl-9-hydroxyellipticinium acetate (Celiptium) are revealed by the detection of thiol-conjugate metabolites in man and rat urine. Besides the unchanged drug and its glucuronide, the cysteinyl- (in man) and the N-acetylcysteinyl- (in man and rat) conjugates have been unambiguously characterized using NMR, UV and mass spectral data. The urinary excretion profile exhibits total excreted products of 21% (in man) and 9% (in rat) with respect to the administered dose. The unchanged drug is found to be the major excreted compound from urine in both species (17% in man, 6.3% in rat); whereas the glucuronide (2.6% in man, 1.5% in rat), cysteinyl- (1.3% in man) and N-acetylcysteinyl- (0.2% in man, 1.2% in rat) conjugates represent the minor excreted compounds. The presence of the latter thio-conjugates provides an indirect proof of the in vivo generation of an oxidized intermediate form of the administered drug.

Synthesis and cytotoxic activity of hydroxylated derivatives of olivacine in relation with their biotransformation.

The chemical synthesis of 9-hydroxyolivacine and 7-hydroxyolivacine based on a biomimetic approach is described. These two hydroxylated derivatives have been found as main in vitro metabolites of olivacine after incubation with rat hepatic microsomes. The pretreatment of animals with benzo[a]pyrene caused a large increase in both microsomal hydroxylations, whereas the pretreatment with phenobarbital caused a weak increase, with a preservation of 9-hydroxylation/7-hydroxylation ratio greater than 1 in both cases. The two hydroxyolivacines have been also found as principal in vivo metabolites of olivacine in rat bile as glucuronide and sulfate conjugates. The pretreatment of animals with benzo[a]pyrene reverses the 9-hydroxyolivacine/7-hydroxyolivacine ratio excretion in bile to a value that is less than 1. In both in vitro and in vivo experiments, the free metabolites were identified by HPLC and UV-visible, MS, and 1H NMR spectra. Hydroxylation at position 9 increases the in vitro cytotoxicity against leukemia L1210 cells (ID50 = 0.06 microM compared to 2.03 microM for olivacine) and an opposite effect is observed for hydroxylation at position 7 (ID50 = 12.8 microM). On the other hand, hydroxylation at position 9 has no effect on the in vivo antitumor activity against L1210. This might be related to the oxidative and conjugative metabolic pathways that play an important role in antitumor activity and deactivation of olivacine and its hydroxy metabolites.

Identification of the glucuronide and glutathione conjugates of the antitumor drug N2-methyl-9-hydroxyellipticinium acetate (Celiptium). Comparative disposition of this drug with its olivacinium isomer in rat bile.

Two metabolites of the antitumor drug N-2-methyl-9-hydroxyellipticinium acetate (Celiptium) were isolated from bile of 10 mg/kg iv-treated rats. These metabolites were characterized by UV-visible, mass and proton-NMR data and identified as (i) 9-(O)-glucuronide conjugate and (ii) 10-(S)-glutathione adduct. Evidence for the same type of metabolites for the isomer N-2-methyl-9-hydroxyolivacinium acetate was supported by HPLC data. Despite their minor structural differences (change into a methyl position), the biliary excretion profile of these two drugs is rather different quantitatively: total metabolites excreted within a 12-hr period is 33 +/- 4% of the administered dose for ellipticinium and 70 +/- 6% for olivacinium. The major metabolite is the 9-(O)-glucuronide conjugate for those two drugs: 25 +/- 3% of the dose of ellipticinium and 67 +/- 5% for olivacinium derivative. In both cases, the presence of the 10-(S)-glutathione adduct as a minor metabolite [1.5 +/- 0.5 of the dose (ellipticinium) and 1 +/- 0.5 (olivacinium)] supports the hypothesis that a "biooxidative alkylation" process could occur in vivo.