Simultaneous determination of dexamethasone and 6 beta-hydroxydexamethasone in urine using solid-phase extraction and liquid chromatography: applications to in vivo measurement of cytochrome P450 3A4 activity.

It has been demonstrated that the formation of the hydrophilic metabolites of dexamethasone, 6 alpha- and 6 beta-hydroxydexamethasone, correlated with cytochrome P450 (CYP) 3A4 enzyme levels. So, the 6 beta-hydroxydexamethasone/dexamethasone urinary ratio could be a specific marker for human CYP3A4 activity. We have developed a sensitive and specific high-performance liquid chromatographic method for the simultaneous quantification of urinary free dexamethasone and 6 beta-hydroxydexamethasone using 6 alpha-methylprednisolone as internal standard. This method involved a solid phase extraction of the three compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate (2 ml) followed by diethyl ether (1 ml). Separation of the three analytes was achieved within 24 min using a reversed-phase Nova-Pak C(18) analytical column (4 microm, 300 mm x 3.9 mm i.d.). An ultraviolet detector operated at 245 nm was used with a linear response observed from 10 to 100 ng/ml for dexamethasone and from 25 to 1000 ng/ml for 6 beta-hydroxydexamethasone. Obtained from the method validation, inter-assay precision was below 15% and accuracy ranged from 95.7 to 110%. The extraction efficiency of the assay was approximately of 99% and was constant across the calibration range. The lower limit of quantitation was 10 ng/ml for dexamethasone and 25 ng/ml for 6 beta-hydroxydexamethasone; at these levels, precision was below 16% and accuracy was 99-109%. This method was applied to in vivo measure of the CYP3A4 activity.

Antiproliferative, differentiating and apoptotic effects elicited by imidazo[1,2-a]pyrazine derivatives.

1. The activity of two series of imidazo[1,2-a]pyrazine derivatives on cell proliferation and differentiation and on apoptosis was examined in relation to their effects on phosphodiesterase (PDE) activity and on purinoceptors. 2. In the first series SC-18 and SC-51 inhibited mitogen-induced 3H-thymidine incorporation in human lymphocytes. 3. The compounds of the new series PAB13, PAB23 and SCA40 inhibited the proliferation of the HEL cell line. 4. Nine imidazo[1,2-a]pyrazine derivatives of the new series have been studied on the Dami cell proliferation. SCA41 and SCA44 inhibited cell growth, SCA40 and PAB40 were moderately effective, whereas PAB12 and PAB30 were devoid of effect. The antiproliferative effects of these six non-cytotoxic compounds could not be related to their action on PDE or on purinoceptors, but rather to their lipophilicity. Conversely, for PAB13, PAB15, and PAB23, the decrease in cell number was related to their cytotoxic and apoptotic effects through their cAMP-increasing and PDE-inhibitory potency, but unrelated to an effect on purinoceptors. 5. Imidazo[1,2-a]pyrazine derivatives decreased the expression of Glycoprotein (GP)Ib in Dami cells while some of them enhanced that of GPIIb/IIIa. These effects appeared to involve inhibition of both cAMP- and cGMP-PDE. 6. These studies demonstrate the potential interest of imidazo[1,2-a]pyrazine derivatives in the query of novel anticancer drugs.

Antiproliferative effects of imidazo[1,2-a]pyrazine derivatives on the Dami cell line.

Since cyclic 3',5'-adenosine monophosphate (cAMP) is involved in cell proliferation and as previous data showed that imidazo[1,2-alpha]pyrazine derivatives (PAB12, PAB30, PAB40, SCA40, SCA41, and SCA44) inhibited cAMP breakdown by a phosphodiesterase (PDE)-inhibitory effect, the aim of the present study was to investigate the effects of these derivatives on proliferation of the Dami cell line in relation with their actions on cAMP content and on PDE isoenzymes isolated from Dami cells. SCA41 and SCA44 inhibited cell growth in a dose-dependent manner, while SCA40 and PAB40 induced a weak inhibition. Growth inhibitions were 40%, 91%, and 60% for SCA41, SCA44 (at 100 microM), and IBMX (at 100 microM), respectively, and could not be related to their effects on cAMP levels. In addition, although all compounds potentiated cAMP formation by prostaglandin E1 (PGE1), no potentiations were observed when the antiproliferative effects of SCA41 and SCA44 were considered. Investigation of derivatives on PDE isoenzymes III, IV, and V indicated non-selective PDE inhibitory effects for SCA41 and SCA44, while SCA40 elicited preferences for type III, and PAB30 and PAB40 preferences for type IV isoenzymes. These effects could not totally explain the antiproliferative activity of the derivatives. The activation of P2 purinoceptors by imidazo[1,2-a]pyrazine did not lead to their antiproliferative effects. Thus, the mechanism of the antiproliferative effects of the compounds remains to be determined. It does, however, depend on the chemical substitutions of the imidazo[1,2-a]pyrazine skeleton and in particular on the 2-carbonitrile presence and the length of the 8-aminoaliphatic group.

Modulation of the megakaryoblastic Dami cell line differentiation by phosphodiesterase inhibitors and imidazo[1,2-a]pyrazine derivatives.

Phosphodiesterase inhibitors have been shown to modulate cell differentiation. We have previously shown that a series of imidazo[1,2-a]pyrazine derivatives displayed inhibitory effects on phosphodiesterase isoenzymes types III. IV and V isolated from Dami cells and on Dami cell growth. In the present study we have investigated the effect of these derivatives on the expression of two differentiation markers, glycoproteins Ib and IIb/IIIa of the human megakaryoblastic leukaemic Dami cell line in comparison to those elicited by 3-isobutyl-1-methylxanthine and selective phosphodiesterase inhibitors of types 1 (8-methoxymetyl-1-methyl-3-(2-methylpropyl) xanthine), III (Milrinone), IV (RO-201724) and V (Zaprinast). Imidazo[1,2-a]pyrazine derivatives, 3-isobutyl-1-methylxanthine and selective phosphodiesterase inhibitors, except 8-methoxymethyl-1-methyl-3-(2-methylpropyl) xanthine, decreased glycoprotein Ib expression. SCA40, SCA41, SCA44 and 3-isobutyl-1-methylxanthine-but not the other compounds affected the expression of glycoprotein IIb/IIIa in a positive manner. The effects of imidazo[1,2-a]pyrazine derivatives on glycoprotein expression appeared to be related to their phosphodiesterase inhibitory potency.

Dissociation between phosphodiesterase inhibition and antiproliferative effects of phosphodiesterase inhibitors on the Dami cell line.

Phosphodiesterase (PDE) inhibitors were shown to inhibit proliferation of various cell types. The present investigation was designed to study the activity of selective PDE inhibitors (8-MeoMIX, milrinone, trequinsin, rolipram, RO-201724, zaprinast, and MY-5445) on the proliferation of the Dami cell line in relation to their effects on cAMP levels and PDE isoenzymes isolated from Dami cells. All compounds, except 8-MeoMIX, elicited antiproliferative effects. Trequinsin, RO-201724, and MY-5445 (100 microM) were found to inhibit cell growth up to 60%, 83%, and 85%, respectively; milrinone, rolipram and zaprinast elicited only weak effects (19-21% at 100 microM). Their growth-inhibitory effects could not be related to their effects on cAMP levels. In addition, although PDE type III and IV inhibitors potentiated cAMP formation due to adenylycyclase activation, no potentiation could be observed when considering their antiproliferative effect. Separation and characterization of PDE of Dami cells revealed the existence of types III, IV, and V isoenzymes. The PDE inhibition found for the PDE inhibitors could not explain their antiproliferative effects. The lack of correlation with cAMP concentrations or PDE inhibition and the high concentrations needed to elicit antiproliferative effects suggest the implication of other parameters, such as cytotoxicity or lipophilicity, or other targets in addition to PDE for the PDE inhibitors tested. Lipophilicity did not seem to be of importance in antiproliferative effects. In contrast, cytotoxic effects, in particular those of trequinsin and MY-5445, could partially explain their negative action on cell growth.

Apoptotic effects of imidazo[1,2-a]pyrazine derivatives in the human Dami cell line.

cAMP-elevating agents like phosphodiesterase inhibitors and purines have been shown to induce apoptosis. In the present work we have studied the effects of imidazo[1,2-a]pyrazine derivatives with a purine-like structure: PAB13 (6-bromo-8-(methylamino)imidazo[1,2-a] pyrazine), PAB15 (6-bromo-8-(ethylamino)imidazo[1,2-a]pyrazine), PAB23 (3-bromo-8-(methylamino)imidazo[1,2-a]pyrazine) on the growth of the Dami cell line in comparison to that of adenosine. The growth effect of PAB13, PAB15 and PAB23 was investigated in relation to their phosphodiesterase-inhibitory action and their activity on purinoceptors. Inhibition in cell growth was up to 71.0%, 76.3% and 89.7% for PAB23, PAB13 and PAB15, respectively and 100% for adenosine. Cell viability was affected in a concentration-dependent manner by PAB13, PAB15 and adenosine, with a correlation between growth inhibition and cytotoxicity. These effects of imidazo[1,2-a]pyrazine derivatives were found to be unrelated to an action on purinoceptors, but rather appear quantitatively linked to their ability in inducing apoptosis through their cAMP-increasing and phosphodiesterase-inhibitory potency.