Dose effect activity of ferrocifen-loaded lipid nanocapsules on a 9L-glioma model.

Ferrociphenol (Fc-diOH) is a new molecule belonging to the fast-growing family of organometallic anti-cancer drugs. In a previous study, we showed promising in vivo results obtained after the intratumoural subcutaneous administration of the new drug-carrier system Fc-diOH-LNCs on a 9L-glioma model. To further increase the dose of this lipophilic entity, we have created a series of prodrugs of Fc-diOH. The phenol groups were protected by either an acetyl (Fc-diAc) or by the long fatty-acid chain of a palmitate (Fc-diPal). LNCs loaded with Fc-diOH prodrugs have to be activated in situ by enzymatic hydrolysis. We show here that the protection of diphenol groups with palmitoyl results in the loss of Fc-diOH in vitro activity, probably due to a lack of in situ hydrolysis. On the contrary, protection with an acetate group does not affect the strong, in vitro, antiproliferative effect of ferrocifen-loaded-LNCs neither the reduction of tumour volume observed on an ectopic model, confirming that acetate is easily cleaved by cell hydrolases. Moreover, the cytostatic activity of Fc-diOH-LNCs is confirmed on an orthotopic glioma model since the difference in survival time between the infusion of 0.36 mg/rat Fc-diOH-LNCs and blank LNCs is statistically significant. By using LNCs or Labrafac to carry the drug, a dose-effect ranging from 0.005 to 2.5mg of Fc-diOH per animal can be evidenced.

Evaluation of cytotoxic properties of organometallic ferrocifens on melanocytes, primary and metastatic melanoma cell lines.

Malignant melanoma is one of the most severe forms of skin cancer, and chemotherapeutic agents currently in use are poorly effective in curing the disease. Here we describe the properties of two organometallic ferrocenyl derivatives, ferrocifen (Fc-OH-Tam) and ferrociphenol (Fc-diOH) that show a specific antiproliferative effect on melanoma cells. After a short incubation period, Fc-OH-Tam is highly cytotoxic on melanoma cells but less toxic on melanocytes. Fc-diOH is slightly toxic at a high concentration but no discrepancy is observed between malignant and normal cells. After a long incubation time the latter is highly toxic for malignant cells but not for normal cells while the former was very highly toxic for primary malignant cells and significantly less toxic for normal cells. We also found that oxidative stress is not implicated in the mechanism of cytotoxicity, since both derivatives neither induce reactive oxygen species (ROS) level in melanocytes nor in melanoma cells. Finally, investigation on hair follicle growth revealed that the two organometallic derivatives induced an irreversible ejection of the hair shaft, thus predicting a potential hair loss side effect if used as a chemotherapeutic treatment.

[How fine needle aspiration biopsy make it possible to avoid extensive surgery. A case of abdominopelvic actinomycosis]. / Comment la ponction guidée à l'aiguille fine peut éviter une chirurgie mutilante.

A case of abdominopelvic actinomycosis diagnosed by fine needle aspiration biopsy is reported. The patient, using an intrauterine device for five years, had a pelvic mass infiltrating the left ovary and five hepatic masses. Cytological smears prepared from an ultrasound-guided fine needle aspiration biopsy of one of the hepatic masses, showed actinomycotic granule. In the literature, few cases of abdominopelvic actinomycosis diagnosed by fine needle aspiration biopsy are reported. This affection, able to simulate a neoplasia on clinical and radiological grounds, is usually diagnosed on the histology of the surgical specimen. Ultrasound and computerized tomography allows to guide fine needle into necrotic areas where the probability to meet actinomycotic granules is maximum and so to avoid an extensive surgery.

Synthesis of CpFe(CO)(L) complexes of hydantoin anions (Cp = eta5-C5H5, L = CO, PPh3), and the use of the 5,5-diphenylhydantoin anion complexes as tracers in the nonisotopic immunoassay CMIA of this antiepileptic drug.

As part of our ongoing development of the CMIA nonisotopic immunoassay method, in which the tracers are metal carbonyl complexes and detection is by Fourier transform infrared spectroscopy, we examined the potential use as tracers of the complexes CpFe(CO)2(5,5-diphenylhydantoin) 2d and CpFe(CO)(PPh3)(5, 5-diphenylhydantoin) 3. The present study involved the synthesis of a series of hydantoin complexes (2a-2d), in particular that of the derivative of 5,5-diphenylhydantoin 2d. The structure of 2d was confirmed by X-ray crystallography. The infrared analysis, establishing the position and intensity of the characteristic metal-carbonyl peaks of complexes 2d and 3 in the 1850-2200 cm-1 region, shows that measurement of the absorbance values of these characteristic peaks will permit quantitative analysis in the picomole range, the norm for routine use in immunoassay and thus suitable for use as CMIA tracers. Cross-reaction rates of these tracers with anti-DPH specific antibodies show that 2d and 3 are both recognized by anti-DPH antibodies (cross-reaction rates 43 and 20%, respectively). In developing a CMIA of DPH with these tracers, it was found that 3, with a single, intense band at 1977 cm-1, had very promising IR characteristics for use in multiassay CMIA, but probably owing to its relatively weak affinity for the antibodies, it was not possible to develop a CMIA for DPH using this tracer. Complex 2d, however, showed better recognition by the antibodies, and using this complex as a tracer, it was possible to develop a particularly sensitive monoassay of DPH by the CMIA method.

Carbonyl metallo immuno assay: a new application for Fourier transform infrared spectroscopy.

We describe here the development of a new, non-isotopic immunological assay termed CMIA (carbonyl metallo immunoassay) that uses metal carbonyl complexes as tracers and Fourier transform infrared spectroscopy (FT-IR) as the detection method. This assay is based on the particular spectral features of these complexes, which show very strong absorption bands in the 1,800-2,200 cm(-1) spectral range where proteins and organic molecules do not absorb. In Section 1, the optimisation of the quantitative detection of these tracers is detailed. In Section 2, the implementation of mono-CMIA is described, including the CMIA assays of three antiepileptic drugs (carbamazepine, phenobarbital, phenytoin). Finally, extension to the simultaneous double- and triple-CMIA of these drugs is reported.

Oxacillin-hydrolyzing beta-lactamase involved in resistance to imipenem in Acinetobacter baumannii.

Acinetobacter baumannii strain A148, a clinical isolate resistant to imipenem (MIC = 32 mg l-1), synthesized two beta-lactamases with pIs 6.3 and > 9.2. The pI 6.3 enzyme hydrolyzed the penicillins, including isoxazoylpenicillins, first-, second- and, to a lesser extent, third-generation cephalosporins. It was inhibited by chloride ions and by the penem beta-lactamase inhibitor BRL 42715. Clavulanate was a weak inhibitor and EDTA did not affect the beta-lactamase activity. This enzyme also hydrolyzed imipenem with a catalytic efficiency (Kcat/Km) of 1500 mM-1 s-1. Moreover, this purified beta-lactamase produced a positive microbiological clover-leaf test with imipenem. Therefore, the pI 6.3 beta-lactamase was considered to be involved in the imipenem resistance of A. baumannii strain A148.

Quantitative analysis of mixtures of metal-carbonyl complexes by Fourier-transform infrared spectroscopy: application to the simultaneous double immunoassay of antiepileptic drugs by the nonisotopic carbonyl metalloimmunoassay method.

The feasibility of a double immunoassay of haptens by the nonisotopic carbonyl metalloimmunoassay (CMIA) method is demonstrated. Three different pairings of antiepileptic medications from the groups carbamazepine, diphenylhydantoin, and phenobarbital (for each of which a mono-CMIA is already available) were assayed by double CMIA. The assay method employs as tracers metal-carbonyl complexes that give very strong signals in the range of 1850-2200 cm-1 in the infrared spectrum, permitting quantitative analysis by Fourier-transform infrared spectroscopy. The fact that the signals are individually assignable and of comparable intensity permits quantitative analysis of mixtures of two tracers. The analysis may proceed in one of two ways: in the simpler case, there is no peak overlap with the two tracers and the quantitative analysis can be performed by simply measuring the absorbance of characteristic peaks of the two tracers. In the second case, in which there is partial or total overlap of peaks, a stepwise calculation provides rapid quantification of the two tracers. These findings allowed us to perform the double CMIA of two antiepileptics in which experimental conditions and time of analysis were strictly identical to those for mono-CMIA. We show here that there is a very good correlation between the results obtained in mono- and double-immunoassay by the CMIA method (correlation coefficient > 0.990).

Production of specific antibodies and development of a non-isotopic immunoassay for carbamazepine by the carbonyl metallo-immunoassay (CMIA) method.

As part of our ongoing work to extend the range of applications of the non-isotopic carbonyl metalloimmunoassay (CMIA), previously developed in our laboratory, we describe here the first CMIA study of carbamazepine. The CMIA method uses a metal carbonyl complex as a non-isotopic tracer, and in this case we chose to employ the dicobalt hexacarbonyl moiety (Co2(CO)6) attached to an alkyne. Two organometallic tracers, 3 and 7, were synthesized, differentiated by the nature and length of the spacer arm of the Co2(CO)6 moiety. Two different coupling methods were subsequently used to synthesize the immunogens 1 and 2, the first one used a carbodiimide, while the second, employed dimethyl adipimidate as coupling agent. Titer values of the antisera obtained by injection of these immunogens into rabbits, were determined by CMIA, using one of the organometallic complexes, 3 or 7, as tracer. Both antisera had higher titer values with the long-chain tracer, 7, than with the short-chain tracer, 3. However these titer values were very different: low for antiserum 1 and high for antiserum 2. The cross-reactivity of antiserum 2 with other antiepileptic drugs was negligible. For competition curves, there was good sensitivity with the antibody 2/3 pairing, while a broad assay range was obtained with antibody 2/7 pairing. These results demonstrate the viability of CMIA as an immunoassay method for carbamazepine, and open the way to development of a simultaneous multiassay by CMIA of the principal antiepileptic drugs.

New applications of carbonylmetalloimmunoassay (CMIA): a non-radioisotopic approach to cortisol assay.

A non-isotopic heterogeneous competitive immunoassay procedure, carbonylmetalloimmunoassay (CMIA) has been applied to the assay of cortisol. The organometallic tracers employed were two stereoisomers (Z and E) of certain cobalt carbonyl complexes of cortisol which have strong, characteristic v(CO) absorptions in the infrared, detectable by Fourier transform infrared (FT-IR) spectroscopy at the femtomole level. The separation of the free and bound organometallic-labelled fractions was achieved by solvent extraction with isopropyl ether. Complete characterization (i.e., dilution, competition and standard curves) of two different polyclonal anticortisol antibodies was possible using the CMIA method. Identical titre values were obtained for the two different stereoisomers used as tracers. In terms of the standard curves, however, the isomers behaved differently depending on which batch of antibody was used. When the best antibody/tracer pair (30 pmol of E isomer; anticortisol 1 antibody) was employed, we obtained a B/B(o)value at 50% of 42 +/- 2.12 pmol and a coefficient of variation of 5%. Finally, preliminary results of a CMIA analysis of plasma cortisol from a patient indicated that reliable and reproducible assays are possible for amounts as small as 50 microliters of serum.

Application of the non-radioisotopic carbonyl metalloimmunoassay (CMIA) to diphenylhydantoin.

As part of our ongoing work to develop the new non-isotopic assay method carbonyl metalloimmunoassay (CMIA), whose efficacy has already been proven in the laboratory for phenobarbital and cortisol, we here present the steps involved in establishing CMIA of 5,5 diphenylhydantoin (DPH), one of the most commonly used antiepileptic medications. First, anti-DPH antibodies were obtained by injection of the immunogen DPH-3-valerate-BSA into rabbits. The titer value and specificity of these antibodies were examined by RIA using [14C]-DPH as tracer, and an antibody batch selected for its high titer value and good specificity for metabolites of DPH and other antiepileptic drugs. Next the organometallic complex Cr(CO)3-DPH, chosen as the CMIA tracer, was synthesized and shown to conserve a high recognition value for anti-DPH antibodies (CR = 200%). Isopropyl ether was selected as the best organic solvent for use in separating the free and bound fractions of the tracer. Employing the Cr(CO)3-DPH complex as tracer and FT-IR spectroscopy as the detection method, we were able to obtain a titration curve by CMIA using an amount of tracer identical to that used in RIA. The titer value obtained in CMIA is approximately twice that obtained by RIA. These results demonstrate the feasibility of DPH assay by the CMIA method.

Synthesis of cobalt carbonyl complexes of cortisol and testosterone. Study of their recognition by specific polyclonal antibodies.

We have synthesized organometallic complexes of steroids (cortisol, testosterone, dihydrotestosterone) for potential use as tracers in nonisotopic carbonyl-metal immunoassays (CMIA). An ethynyl/CO2(CO)6 fragment at the end of a five-atom spacer was coupled to position 3 of the steroid skeleton. In the case of cortisol, we exploited the difference in reactivity of the ketone and enone functions toward amines in order to form an enamine which was then made to react with carboxymethylamine to yield 3-[(carboxymethyl)oxime] steroid. Activation of the carboxylic acid function with N,N'-dicyclohexylcarbodiimide in the presence of propargylamine introduced an acetylenic function at the end of the spacer. The triple bond was then complexed by CO2(CO)8 to form complexes 5a-c. Complexes for use in CMIA should be stable in biologic media and effectively recognize specific antibodies. Complexes 5a-c were stable in the buffers we use in biochemical tests. Their cross reactivities for anti-cortisol and anti-testosterone antibodies ranged from 50 to 110% according to batch, indicating, first, that the addition of an organometallic complex in position 3 of the steroid skeleton does not hinder recognition between the organometallic steroid and antibody and, second, that their individual behavior differs substantially according to antibody batch. Although all of these complexes could be used as tracers in CMIA, it is necessary, in each case, to establish which tracer-antibody duo gives rise to the most sensitive immunoassay.

Use of Fourier transform infrared spectroscopy for the simultaneous quantitative detection of metal carbonyl tracers suitable for multilabel immunoassays.

We describe here a new approach for multilabel immunoassays. The starting point of this approach is a new nonradioisotopic immunoassay (carbonyl metallo immunoassay) based on the use of metal carbonyl complexes as tracers and Fourier transform infrared (FT-ir) spectroscopy as the detection method. We show here that the judicious choice of the organometallic tracers associated with multicomponent analysis of FT-ir spectroscopic data provides the simultaneous rapid and reliable quantitation of pmol of the organometallic tracers. The feasibility of this approach is demonstrated in the case of the two major antiepileptic drugs phenobarbital and carbamazepine.

Syntheses and affinities of novel organometallic-labeled estradiol derivatives: a structure-affinity relationship.

Two series of novel estradiol derivatives, including cationic species, labeled with organometallic fragments Cr(CO)3, Cp*Ru+, or Cp*Rh2+ [Cp* = eta 5-C5(CH3)5] either in the 17 alpha-position or on the A-ring were synthesized, and their relative binding affinities (RBA) for the estradiol receptor were determined. The Ru(II) and the Rh(III) cationic derivatives were obtained as stable salts with the following counter anions (BF4-, PF6-, CF3SO3-). The satisfactory RBA values obtained for most complexes belonging to the 17 alpha series confirm that this position tolerates the presence of bulky neutral species. For instance, complex 4, in which the organometallic fragment Cr(CO)3 was attached to the phenyl ring of the 17 alpha-phenylethynyl fragment, exhibited an RBA value of 24%, very similar to that of the uncomplexed estrogen derivative 3. Surprisingly, the analogous cationic species 6 had no affinity for the estradiol receptor. This unprecedented result shows that the hormone binding site of the estrogen receptor does not tolerate the presence of a positive charge in the 17 alpha-position of the steroid. On the other hand, the alpha-face of the A-ring of estradiol did tolerate positively charged organometallic fragments bearing bulky substituents although the RBA value tended to decrease with increasing charge. The counterion in these cationic derivatives also affected binding affinity. For instance, the Ru(II) species 7a containing an CF3SO3- ion exhibited a reasonable RBA value (5.8%) compared to analogous species 13 with a PF6- ion (RBA of only 0.1%). Moreover, the triflate counteranion preserved the phenolic form of the A-ring of the estrogen derivative whereas the PF6- derivative was unstable and rapidly converted into the dienonylic form in buffer. The compared RBAs of the neutral and cationic species illustrate the preferences of the receptor hormone binding site in accepting or rejecting species of hydrophobic or hydrophilic character.

Carbonylmetalloimmunoassay (CMIA) a new type of non-radioisotopic immunoassay. Principles and application to phenobarbital assay.

A new non-radioisotopic immunoassay procedure, which we have termed carbonylmetalloimmunoassay (CMIA), is described. The tracers used in this approach are organometallic carbonyl complexes that can be detected at femtomole levels (300-700 fmol) by Fourier transform infrared (FT-IR) spectroscopy. The validity of the technique has been tested in a phenobarbital assay using as the marker a cyclopentadienylmanganese (I) tricarbonyl (cymantrene) moiety, ethyl acetate extraction to separate the free and bound organometallic fractions, and FT-IR spectroscopy to detect the CO stretching modes of the organometallic label. Typical dilution and standard curves obtained with this CMIA procedure are presented. The method was of comparable sensitivity to a [14C] radioimmunoassay (RIA) for the detection of phenobarbital. A comparison of the results for phenobarbital assays by both CMIA and RIA showed that higher titres were obtained using the CMIA method. The standard curves suggest that CMIA is a reliable and reproducible immunoassay procedure for phenobarbital.

Fourier transform infrared spectroscopic method for the quantitative trace analysis of transition-metal carbonyl-labeled bioligands.

A quantitative FT-IR spectroscopic method has been developed for the trace analysis in chlorinated organic solvents of transition-metal carbonyl-labeled bioligands. In order to illustrate the widespread analytical potential of the method, three derivatives of the female hormonal steroid 17 beta-estradiol, containing Cr(CO)3, Cp2Mo2(CO)4 (Cp = eta 5-C5H5), and Co2(CO)6 as labels, and the anticonvulsant drug phenobarbital, labeled with (eta 5-C5H4)Mn(CO)3, were examined. The cobalt carbonyl marker proved to be the best sulted for quantitative analysis purposes, and the minimum tracer quantity detectable for this particular marker (64 scans, 4-cm-1 resolution, 3.5 min) was optimized in CCl4 solution at about 300 fmol (or 0.3 pmol, 180 pg) by using an ultralow volume (23.0 microL), gold light-pipe IR solution cell and a liquid nitrogen cooled, InSb (indium antimonide) IR detector. The repeatability of this radically different analytical procedure over the concentration range 1.0 x 10(-6) to 5.0 x 10(-8) M was good (coefficient of variance less than or equal to 6%) and the method provides the basis for a new immunological test--carbonylmetalloimmunoassay (CMIA).

(N-succinimidyl 4-pentynoate)hexacarbonyldicobalt: a transition-metal carbonyl complex having similar uses to the Bolton-Hunter reagent.

The synthesis of the transition-metal carbonyl complex (N-succinimidyl 4-pentynoate)hexacarbonyldicobalt [[(C4H4O2N)O(CO)CH2CH2C identical to CH]Co2(CO)6] is described. This cobalt carbonyl complex is structurally similar to the Bolton-Hunter conjugation reagent and has been successfully employed as a nonradioactive tracer for labeling the drug carbamazepine. The metal carbonyl tracer can be detected at extremely low concentrations (ca. 1 pmol) by FT-IR spectroscopy in the v(CO) region (2150-1800 cm-1). The cobalt carbonyl labeled carbamazepine retains good recognition for anti-carbamazepine antibodies. This novel labeling procedure, which can be broadly termed carbonylmetalloimmunoassay (CMIA), has considerable potential for assaying a wide range of biological materials.

Evidence for broken minocycline by NMR and HPLC techniques: a new additional resistance mechanism mediated by tetB determinant.

As demonstrated by microbiological assays, a decrease in the active minocycline level occurs in spent media from each Escherichia coli K12 recipient containing one of 10 different plasmids bearing tetB determinants. No such decrease was detected when tetA, C, D or E determinants were tested under the same conditions. Likewise, no decrease in tetracycline or doxycycline levels was detected when 20 plasmids bearing tetA to E determinants were tested. Studies carried out by nuclear magnetic resonance and high pressure liquid chromatography proved that minocycline is broken by a mechanism mediated by the tetB determinant. This new mechanism can be considered as additional to the active efflux of minocycline.

Organometallic derivatives of estradiol as bioligands: targetted binding of the estradiol receptor.

The complexation of estrogens by transitional metal units e.g. (alkyne)Co2(CO)6 and (alkyne)Mo2Cp2(CO)4, at the 17 alpha-position brings about a dramatic change in the chemical behavior of these compounds with respect to that of the free ligands. The 17 beta-OH function becomes particularly labile, even in weakly acidic medium, giving rise to carbenium ion-like species, from which, depending on the metal and the nucleophile, substitution, elimination and rearrangement take place. This situation provides the basis for a new type of active site directed-reagent for estradiol receptor. The hypothesis of vicinal space positioning of an acidic and a nucleophilic group in the estradiol receptor cavity is examined in the light of the amino-acid composition of the steroid binding domain. The requirement of the sulfhydryl group of a cysteine residue is suspected in the first step of the receptor inactivation process.

Organometallic estrogens: synthesis, interaction with lamb uterine estrogen receptor, and detection by infrared spectroscopy.

As an integral part of the development of a new technique using organometallic markers for the detection of hormone receptors by FT-IR spectroscopy, a series of estradiol derivatives labeled with Cr(CO)3 or Cr(CO)2CS fragments on the A ring has been synthesized. The stereochemistry of one of these steroids, alpha-[3-(dimethyl-tert-butylsiloxy)-17 beta-estradiol]dicarbonyl(thiocarbonyl)chromium(0), has been established by X-ray diffraction. The organochromium-labeled steroids are stable in aqueous methanol solution, and their relative binding affinities to estrogen receptor have been determined; these values vary from 0.4 to 28%. The complex exhibiting the strongest affinity, [3-O-(3-hydroxypropyl)-17 beta-estradiol]-chromium tricarbonyl complex, has been prepared in a tritiated form with a high specific activity (4.1 Ci/mmol). This tritiated hormone binds reversibly to the estradiol receptor in lamb uterine cytosol with an affinity (Kd = 0.85 nM) and number of binding sites (n = 770 fmol/mg of protein) close to the values observed for estradiol itself. The level of nonspecific binding is low, and the hormone is not bound significantly to other nontarget tissues. The observation that the binding affinity of the steroid depends on which side of the steroidal A ring the organometallic label is bound demonstrates the nonequivalence of the two sides of the A ring with respect to the receptor site. The FT-IR spectra of the organochromium markers in the v(CO) region can be used for the detection of the estradiol receptor in lamb uterine cytosol.