Danièle Duval

Quantification of plasma and intracellular levels of the HIV protease inhibitor ritonavir by competitive ELISA

The HIV protease inhibitor ritonavir (Norvir; ABT-578), currently used in combination with nucleoside analogs and other protease inhibitors in anti-HIV therapy, has previously been quantified by an HPLC procedure. Here, we report the first convenient one-step competitive ELISA for measuring plasma and intracellular ritonavir in HIV patients. Anti-ritonavir antibody was raised in rabbits using ritonavir-KLH conjugate as immunogen, and the enzymatic tracer was prepared by coupling the drug to acetylcholine esterase. Samples for analysis were first extracted with methanol. Bound/free separation was achieved in a microtiter plate previously coated with anti rabbit IgG monoclonal antibody. Fifty percent inhibition was observed at 1 ng/ml ritonavir and the method accurately and specifically detected as little as 3-4 ng/ml of plasma ritonavir as well as intracellular drug in the peripheral blood mononuclear cells of patients undergoing ritonavir therapy. Within-run and day to day coefficients of variation were below 10% and the drugs currently used in HIV therapy did not interfere with the test. The ELISA was applied to the measurement of plasma ritonavir and to the determination of the extracellular/intracellular drug level ratios in HIV patients receiving long-term multidrug therapy.

Preparation of dehydroepiandrosterone, testosterone and progesterone antigens through 7-carboxymethyl derivatives: Characteristics of the antisera to testosterone and progesterone

Dehydroepiandrosterone, testosterone and progesterone 7-carboxymethyl derivatives were prepared: 7 alpha and 7 beta epimers were separated and coupled to bovine serum albumin. Preliminary studies of the antisera induced by these antigens showed that they have high affinity and good specificity.

Four steroidal alkaloids from the leaves of Buxus sempervirens

Four new steroidal alkaloids, N20-formylbuxaminol E [(20S)-16alpha-hydroxy-20-(formylamino)-3beta-(dimethylamino)-9,10 -seco-buxa-9(11),10(19)-diene] (1), O16-syringylbuxaminol E [(20S)-16alpha-syringoyl-3beta-(dimethylamino)-20-(amino)-9, 10-seco-buxa-9(11),10(19)-diene] (2), N20-acetylbuxamine G [(20S)-20-(acetylamino)-3beta-(methylamino)-9,10-seco-buxa-9(11),1 0(19)-diene] (3) and N20-acetylbuxamine E [(20S)-20-(acetylamino)-3beta-(dimethylamino)-9,10-seco-buxa-9(11) ,10(19)-diene] (4) were isolated from the leaves of Buxus sempervirens. Their structures were determined mainly on the basis of 2D NMR studies.

Quantification of Lamivudine (3TC) by Competitive Immunoassay

Lamivudine or 3TC, the (-) eniantiomer of 2-deoxy-3-thiacytidine, is a prototype of a novel class of levogyre dideoxynucleosides analogues used in treatment of HIV and HBV infection. We describe a method corresponding to the first enzyme immunoassay for quantifying this antiviral drug. This technique use an enzyme conjugate that not require the use of radioactive labelling. In this study, anti-3TC antibodies were raised in rabbits by immunising with 3TC-HS-kelhoyle limpet hemocyanin (KLH) conjugate.

Design, synthesis and studies of triphosphate analogues for the production of anti AZT-TP antibodies.

Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular modelling, (ii) their synthesis and (iii) their use for producing anti AZT-TP antibodies in the aim of developing an immunoassay for therapeutic drug monitoring.

Synthesis of 7α- and 7β-carboxymethyl-9(11)-ene derivatives of estrone and estradiol

Abstract As part of a search for derivatives designed to conjugate to amino groups, either of a protein carrier for antibody production or of an immobilized side-chain on a polymer for affinity chromatography, functionalized estrone and estradiol analogues were prepared. These modified steroids were obtained via the introduction of a carboxymethyl side-chain at the C-7α and C-7β position on an adrenosterone derivative and were then aromatized on the A ring. These new compounds are unsaturated at the C-9 (11) position, which could be useful for a second modification.

Design and Synthesis of Nucleoside Phosphate Mono(Aminoalkyl)Ester

Abstract This report describes the one step synthesis of nucleosides monophosphates alkylamines via direct condensation of N-protected phosphate alkylamines on nucleosides, providing a method for the preparation of nucleotide haptens.