Stability parameter estimation at ambient temperature from studies at elevated temperatures.

The determination of specific kinetic constants k(i) in pH-profile studies is often undertaken at ambient temperature. However, when dealing with a drug substance that is stable at ambient temperature, the pH-profile study is conducted at a chosen elevated temperature and the kinetic parameters are given at this particular elevated temperature. But in stability studies we generally need kinetic constants at ambient or storage temperature for practical reasons (information and storage conditions of formulation). To assess this ambient kinetic information from studies at elevated temperatures, cumulative sequential steps are usually employed with very few statistical concerns on the final estimates. The statistical problems on these final estimates in cumulative procedures are highlighted in many papers. Because these stability parameters are useful for drug formulation and storage conditions, good practical decisions have to be made on the basis of statistically unbiased identified parameters. We propose in this paper a nonlinear model that allows the direct determination of specific activation energies E(ai) that are linked to the specific kinetic constants k(i). Hence, a mathematical relationship between drug concentration C, pH, temperature T, and time t is obtained. Kinetic data from acetylsalicylic acid (ASA) hydrolysis (first-order kinetics) are used to validate the model. The results show that it is possible to obtain directly, by an extrapolation procedure, the kinetic parameters (specific kinetic constants k(i), specific activation energies E(ai), and dissociation constant pK(a)) at low temperature from data gathered at elevated temperatures using more meaningful statistics.

Photodynamic DNA damage mediated by delta-aminolevulinic acid-induced porphyrins.

The mutagenic properties of UVA are thought to be predominantly radical-mediated, which supposes endogenous sensitizers. In order to investigate a possible role of porphyrins, their synthesis was induced in a murine leukemia P388D1 cell model by treatment with delta-aminolevulinic acid (delta-ala). Intra-cellular protoporphyrin IX reached a plateau after about 2 h, whereas soluble porphyrins, probably the photostable uro- and/or coproporphyrins, were excreted. Irradiation of treated cells by UVA (tanning lamp) but also by visible light was found to generate in DNA a significant increase of 8-oxo-7,8-dihydro-2'-deoxyguanosine, a mutagenic marker of oxidative damage. The different parameters involved in this photodynamic effect are reported, namely delta-ala concentration and loading time, light dosage and the influence of intracellular and medium-excreted porphyrins. These results point to an implication of porphyrins in solar-induced carcinogenicity but also in possible adverse effects of the medical applications of photodynamic therapy and diagnosis.

Application to drug-food interactions of living cells as in vitro model expressing cytochrome P450 activity: enzyme inhibition by lemon juice.

Classical inhibitors of human cytochrome P450 3A4 activity, such as ketoconazol and quercetin, are tested to prove the efficiency of a new metabolisation model using living entire cells. Grapefruit juice is a well-known potent inhibitor of cytochrome P450 3A4 activity. With regard to the clinical relevance of grapefruit juice-drug interactions, an investigation of other common juices is undertaken with this in vitro model. The CYP3A4 activity is measured by the formation of the 6beta-hydroxytestosterone, which is quantified by an isocratic high performance liquid chromatography. It is demonstrated for the first time that lemon juice significantly inhibits by 60+/-3% the CYP3A4-mediated oxidation. Grapefruit juice inhibits this activity by 82+/-4%. The mechanism of lemon juice inhibition is competitive, whereas it is mixed for grapefruit juice. These results suggest that our in vitro model combined with our analytical method is applicable for the investigation of the inhibition of CYP3A4 not only by chemical inhibitors but also by natural food products.

Chromatographic determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA: a validation study.

Although a series of biomarkers are widely used for the estimation of oxidative damage to biomolecules, validations of the analytical methods have seldom been presented. Formal validation, that is the study of the analytical performances of a method, is however recognized as the best safeguard against the generation and publication of data with low reliability. Classical validation parameters were investigated for the determination of an oxidative stress biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in cellular DNA, by high-performance liquid chromatography coupled to amperometric detection (HPLC-EC); this modified base is increasingly considered as a marker of oxidative damage to DNA, but many questions are still raised on the analytical methods in use. Upon a rigorous statistical evaluation of the quality criteria currently required for assays in biological media, including selectivity, linearity, accuracy, repeatability, sensitivity, limits of detection and quantification, ruggedness and storage at different stop points in the procedure, the HPLC-EC assay method is found mostly reliable. The present validation attempt demonstrates that (i) the HPLC-EC assay of 8-oxo-dG provides consistent data allowing to reliably detect an increase of this biomarker in cellular DNA; (ii) a harsh oxidative stress does not hinder the enzymatic digestion of DNA by nuclease P1; and (iii) the analytical results must be expressed relative to the internal standard dG which significantly improves both repeatability and sensitivity. Whereas the described assay minimizes the artifactual production of the analyte from processing and storage, this cannot be totally ruled out; the true 8-oxo-dG base levels still lack a definitive assay method, which remains a considerable analytical challenge and the object of controversy.

Improved kinetic parameter estimation in pH-profile data treatment.

Statistical problems in temperature stability parameter estimation have been the subject of many papers whereas statistics in, pH-profile parameter estimation have focused little attention. However, the conventional two step method used in data treatment in both cases leads to identical statistical problems. The aim of this study is then to introduce a method that improves statistics in pH-profile parameter estimation. A one step non-linear method that takes into account the errors in drug content determination is proposed. A mathematical relationship between drug content C, pH and time t is tested. The proposed method allows the estimation of the specific kinetic constants and the dissociation constant (pK(a)) in a single run. The most likely experimental initial drug contents C(0j),. where j is the index of a given experiment, are also determined. This approach that takes into account all relevant experimental information for the estimation of kinetic parameters is more rigorous from a statistical viewpoint than the classical two step methods. Kinetic data from acetylsalicylic acid (ASA) hydrolysis was used for the tests.

Incorporating batch effects in the estimation of drug stability parameters using an Arrhenius model.

The nonlinear estimation of drug stability parameters (energy of activation Ea and shelf-life tY) by conventional approaches employs equations relating drug content determination C at time t and temperature T. The identification procedures lead to the determination of only one initial drug content C0 for several different experiments. However, it is well known that because of experimental concentration variation or of intentional modification of the experimental schedule, there are as many initial drug contents as experiments. For these reasons, a method which takes into account batch effects is proposed to determine stability parameters and also all initial drug contents C0j where j is the index of experiment in one step. This method is more accurate from a statistical viewpoint and is suitable for data treatment in pharmaceutical industries where the initial drug content of each batch entering the stability program can be checked a posteriori. The application of this method is shown on real kinetic data from the hydrolysis of acetylsalicylic acid (ASA).

Multidrug resistance modifies polyamines uptake in P388 murine lymphoma cells: experimental and modeling approach.

Polyamines (putrescine, spermidine and spermine) are ubiquitous compounds, essential for cell growth. This paper compares the polyamine transport between sensitive P388 murine lymphoma cells and two multidrug resistant P388 sublines with the assistance of an experimental model. This new model allows the characterisation of the whole polyamines uptake and efflux. Three parameters are identified by the model: two rate constants (K+ for the uptake and K- for the efflux) which are considered as physical constants specific to the transport of one polyamine in one cell type, and Ci(o) which represents the initial intracellular concentration. This model well describes our experimental results of polyamine transport across the P388 cell plasma membrane. Multidrug resistant P388 cells exhibit spermine uptake significantly higher than that of sensitive cells when on the opposite, putrescine enters more rapidly into the sensitive P388 cells. In conclusion, comparison of polyamine transport between sensitive and multidrug resistant P388 phenotypes shows large and significant differences.

Simple and rapid enzymatic assay of ornithine decarboxylase activity.

Since the induction of putrescine synthesis by ornithine decarboxylase (ODC) is observed in many pathological and physiological processes, a useful and simple method to assay this enzyme activity should be an interesting tool to quantify the biological importance of its induction. An enzymatic method to assay ODC is reported here. This method is based on the reaction between putrescine and soya diamine oxidase. The reaction releases H(2)O(2), which is measured by a colorimetric method. The validation of this method showed good accuracy (98+/-5% of recovery). High precision and reproducibility were obtained. A linearity with a correlation coefficient of 0.999 in the range of 2.5-25 nmol was obtained. This method is also rugged and specific. The application of the assay of ODC activity showed that it is useful as a rapid and simple tool for assaying ODC activity in vitro. Comparison with the HPLC determination of ODC activity shows strong correlation along with the high accuracy of the two methods.

Inhibition of ornithine decarboxylase by ifenprodil.

Ifenprodil (NMDA receptor antagonist) was tested as an inhibitor of ornithine decarboxylase. It was found that ifenprodil inhibited ornithine decarboxylase activity with the same potency as alpha-difluoromethylornithine, a major inhibitor of ornithine decarboxylase. This result suggests that ifenprodil could target either the polyamine site on the NMDA receptor complex or/and polyamine biosynthesis.

Ethyl acetate extraction procedure and isocratic high-performance liquid chromatographic assay for testosterone metabolites in cell microsomes.

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for separation of testosterone and its main metabolites over the nominal range 20 to 40 microg/ml and 280 to 4600 ng/ml, respectively. Mobile phase composition (phosphate buffer-methanol-acetonitrile, 50:38.5:11.5) was optimised by studying the influence of numerous chromatographic parameters. The most critical one was the ratio CH3CN/CH3OH. Good recoveries (around 90% for all compounds) and an improved specificity were assessed by a double ethyl acetate extraction of biological samples. According to the performance criteria tested, the method could be applied to enzymatic inhibition and induction in vitro studies.

Synthesis and cytotoxicity studies of new analogues of polyamines.

In order to regulate simultaneously the biosynthesis and the transport of natural polyamines, the synthesis of a series of N-methylated analogues of N,N1-Bis(benzyl)-alkanediamines (propanediamine and butanediamine) was achieved and the cytotoxicity of these compounds on the P388D1 cell line was determined. Experiments were conducted in a growth culture medium 20 microM of 2-mercaptoethanol or 0.1 mM of aminoguanidine. Their cytotoxic effects were compared to those obtained under the same conditions with natural polyamines known as toxic compounds at high concentrations. The IC50 of each compound was found very similar for all experimental conditions (IC50 approximately 150 microM) at the opposite of spermidine and spermine which were less toxic (IC50 > 500 microM) when cells were grown in the presence of aminoguanidine (a specific inhibitor of fetal calf serum's PAO). The DL-difluoromethylornithine (DFMO) and MDL 72527DA, two well known inhibitors of ornithine decarboxylase (ODC) and Polyamine Oxidase (PAO) respectively, had no toxicity on the P388D1 cells compared to our compounds. Our most toxic compound was N1,N4-Bis(benzyl)-N1,N4-bis(methyl)-1,4-butanediamine (6) with an IC50 of 127 +/- 3 microM (in culture medium alone). The synthesis of the beta-aminothioether derivative of N-benzylputrescine (11) and the beta-aminothiol derivative of N-benzylspermidine (13) were also related. The Compound 11 was tested against the P388D1 cells, and did not show any cytotoxic effect. The N-methyl derivatives should give the advantage to be used at low concentrations than those used to test the DFMO.

Ability of bisbenzylputrescine and propanediamine analogs to modulate the intracellular polyamine pool of P388D1 cells.

The effects of a series of bisbenzyldiamine analogs have been tested on P388D1 cell line in vitro. Their effects on cell growth, polyamine oxidase (PAO) activity and intracellular polyamine content were determined. The cytotoxicity tests were performed in culture medium supplemented with 100 micromol/L aminoguanidine (I), 100 micromol/L aminoguanidine and 100 micromol/L N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527) (II), and finally 100 micromol/L aminoguanidine and 200 micromol/L D,L-difluoromethylornithine (DFMO) (III). The IC50 values under conditions I and III were similar, suggesting that inhibition of ornithine decarboxylase by DFMO did not affect the biological effect of our derivatives. Spermine and spermidine remained nontoxic in conditions I and III. However in the condition II, the toxicity of all tested compounds (excepted spermidine) was increased, suggesting that the inhibition of cellular PAO increased their toxicity. The enzymatic test of PAO showed that at high doses inhibition of this enzyme by putrescine analogs occurred, while the N-methylated propanediamine derivative increased the enzyme activity; however, these results do not correlate with cytotoxicity tests. When these derivatives were incubated for 48 h with the cells, all of them increased the cell content in putrescine (approximately 160%) and spermine (approximately 145%) and decreased the spermidine content (approximately 75%) without any modification of the total amount of polyamine. The correlation between the cytotoxic results and the intracellular polyamine determination shows that the increase in spermine content along with the inhibition of retroconverting PAO enzyme increases the toxic effect of tested compounds (including spermine), suggesting that spermine toxicity is more important in the absence of intracellular oxidation processes.

Isocratic high-performance liquid chromatographic method for the separation of isradipine and its main metabolites. Application to in vitro metabolization by h3A4/OR cells.

A reversed-phase HPLC method was developed for the study of isradipine oxidation in vitro. The drug and its main metabolites were determined after extraction from culture media and from h3A4/OR cells having the cytochrome P450 isoenzyme involved in the xenobiotic metabolization. The HPLC assay was fully validated and was used to follow the biotransformation kinetics of isradipine.

Evidence of difference between cytotoxicity, cell cycle and morphonuclear effects of polyamines on sensitive and multidrug resistant P388 cell lines.

This paper reports studies on the influence of multidrug resistance (MDR) on the mechanism of polyamine toxicity. The effects of putrescine (PUT), spermidine (SPD) and spermine (SPM) on morphonuclear parameters and cell cycle were studied by means of digital cell image analysis. This reveals that only SPD and SPM condense chromatin inducing a strong decrease in the nuclear area and a cell-cycle arrest in phase G2 in P388/s and the two MDR sublines. A significant difference was observed between the sensitivity of the two phenotypes, which confirms results obtained by means of a microculture tetrazolium test which showed that SPD and SPM were highly, but very differently, cytotoxic on sensitive and MDR sublines, unlike PUT, which was not toxic. This encourages us to study more thoroughly possible differences in polyamine metabolic enzymes and uptake in these cells, to enable us to acquire a better understanding of the impact of MDR phenotype on the polyamine pathway.

Simultaneous determination of cytotoxic (adriamycin, vincristine) and modulator of resistance (verapamil, S 9788) drugs in human cells by high-performance liquid chromatography and ultraviolet detection.

Multidrug resistance (MDR), which was described for structurally and mechanistically unrelated anticancer agents, was modulated in vitro by a series of compounds which were of different chemical origin. In this situation, the selection of a correct assay dosage to study the MDR modulation mechanism was a problem. We developed a high-performance liquid chomatography (HPLC) method which enabled the simultaneous determination of three major cytotoxins (adriamycin, daunorubicin, vincristine) and two well-known modulators (S 9788, verapamil). This assay was fully validated and was used to follow, for the first time, the uptake and accumulation behaviour of adriamycin and S 9788 co-incubated with resistant and sensitive cell lines (KB-3-1; KB-A1).

Naphthylisoquinoline alkaloids exhibit strong growth-inhibiting activities against Plasmodium falciparum and P. berghei in vitro--structure-activity relationships of dioncophylline C.

The growth-inhibiting activities of naturally occurring naphthylisoquinoline alkaloids against asexual blood stages of Plasmodium falciparum (NF 54, clone A1A9) and P. berghei (Anka) were studied in vitro. Three of the alkaloids [7-epi-dioncophylline A (8b), dioncolactone A (4), and 5'-O-demethyl-8-O-methyl-7-epidioncophylline A (11)] displayed good activities against both parasites, with median inhibitory concentrations (IC50) of 1-5 micrograms/ml. Dioncophylline C (2), however, was even better, with IC50 of 0.014 microgram/ml (P. falciparum) and 0.015 microgram/ml (P. berghei) and therefore regarded as a promising lead for studies of structure-activity relationships. The free N- and 8-OH-functions were shown to be prerequisites for the outstanding activity of this molecule against P. falciparum, the presence of at least one free phenolic OH-function appearing to be essential for any activity. Initial experiments with derivatives of ancistrocladine (1) show that, in contrast to 2, N-derivatization of this alkaloid leads to increased activity against P. falciparum.

Cytotoxicity of polyamines to Amoeba proteus: role of polyamine oxidase.

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H2O2, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N1-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N1-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3-20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid (approximately 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity (approximately 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.

Improved high-performance liquid chromatographic method for the determination of polyamines as their benzoylated derivatives: application to P388 cancer cells.

A simple reversed-phase HPLC method was developed for the determination of eight polyamines or monoacetylpolyamines, as their benzoylated derivatives. Interfering products, inherent to the benzoyl chloride derivatization technique (benzoic acid, methyl benzoate and benzoic anhydride), were identified. A new derivatization procedure for their total elimination was developed without any loss of sensitivity and selectivity. Not only the HPLC method was validated, but also the choice of an internal standard was investigated. The results show that it is possible to use this HPLC assay to determine the polyamine content in P388 cancer cells. Furthermore, the method is now being used to evaluate the uptake of various polyamines by P388 cancer cells and by other cancer and parasitic cells.

HPLC determination of a new multidrug resistance modulator (S9788) extracted from cancer cells in vitro.

S9788 is a novel triazinodiaminopiperidine derivative which reverses the multidrug resistance of tumour cells to anticancer drugs. In this study, a new HPLC method was developed to determine this compound in P388 leukaemia cells. The influence of various parameters (composition and pH of the mobile phase, nature of the column) on the separation of S9788 and derivatives was investigated. Using a microsphere C18 column and the optimal mobile phase (acetonitrile-0.4 M phosphate buffer containing 0.2% triethylamine, 40:60 v/v, pH 6.5) it was possible to separate S9788 and seven hypothetical metabolites and derivatives in 15 min. The limits of detection and quantification of S9788 are 75 and 250 pg, respectively. This MDR modulator was extracted from biological media by a rapid two-step procedure which removed proteins before direct injection of the sample. Absolute recoveries ranged from 90 to 100% with a mean RSD (%) lower than 5.

Chlordiazepoxide photoisomerization kinetics into oxaziridine. A HPLC study.

It was proved that the N(4)-oxide group included in chlordiazepoxide (CDZ) is involved in its phototoxicity. At a wavelength of 350 nm, CDZ photoisomerizes only into oxaziridine (OXA) which is not available as standard. In the course of cytotoxicity investigations, the optimal CDZ irradiation conditions were established as acetonitrile as solvent, 10 degrees C as temperature of the irradiated solutions and 70-90 min as irradiation time for solutions in the range of 12.2-152.0 microg/ml. The kinetic parameters of the CDZ photodegradation reaction order have been calculated using an appropriate algorithm. In all cases, the first order reversible or irreversible was selected by Akaïke's criteria. The percentage of undecomposed CDZ and OXA generated after irradiation were determined by a reversed HPLC method. The latter also permitted the separation of CDZ major impurities in aqueous solutions (demoxepam and 2-amino-5-chlorobenzophenone) as well as the oxaziridine of demoxepam. In this study, the experimental irradiation conditions allowed us to produce 98% pure OXA from CDZ. This HPLC method could be easily extended to the analysis of the molecules in pharmaceutical studies.