Biomedical subjects

E Ezan

Publications and source records attributed to E Ezan.

At least 37 records · Page 2Linked to original sources

PubMed1999

In vivo assessment of captopril selectivity of angiotensin I-converting enzyme inhibition: differential inhibition of acetyl-ser-asp-lys-pro and angiotensin I hydrolysis.

Angiotensin I-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood pressure regulation. The demonstration that the hemoregulatory peptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a natural and specific substrate of the N-active site of ACE suggests that this enzyme may have a new physiological role such as the modulation of hematopoietic stem cells. In vitro studies have shown that ACE inhibitors displayed various potencies in inhibiting the degradation of different natural or synthetic substrates of ACE, among which captopril inhibits AcSDKP hydrolysis more potently than angiotensin I hydrolysis. To look for this selectivity in vivo, we investigated the pharmacodynamic effect of increasing doses of captopril (0.01-10 mg/kg) during the 90 min after i.v. administration to spontaneously hypertensive rats. Plasma and urinary AcSDKP levels were measured. The renin-angiotensin system was evaluated by measurements of ACE activity in plasma samples, using the synthetic substrate Hip-His-Leu, by determinations of plasma renin concentrations and measurements of arterial blood pressure. The results showed that captopril (0.01-0.3 mg/kg) selectively inhibited AcSDKP hydrolysis, with limited effects on the renin-angiotensin system. AcSDKP levels in plasma and urine rose to a plateau 4 times the basal level for doses more than 0.3 mg/kg. All of the parameters reflecting the renin-angiotensin system were significantly affected at doses of 1 and 10 mg/kg. The present study therefore confirms that captopril can be used to protect hematopoietic stem cells during antitumor chemotherapy while having only a limited effect on cardiovascular homeostasis.

Angiotensin I↗

PubMed1999

Effect of angiotensin-converting enzyme inhibition on plasma, urine, and tissue concentrations of hemoregulatory peptide acetyl-Ser-Asp-Lys-Pro in rats.

The hemoregulatory peptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been reported to accumulate in plasma and urine after the oral administration of angiotensin-converting enzyme (ACE) inhibitors in humans. It is unknown whether such an accumulation also occurs in tissues. We administered captopril (3, 10, or 30 mg/kg) orally for 2 weeks to Wistar rats. In a second experiment, captopril (10 mg/kg) was administered for 9 days and was followed by a 1-h i.v. infusion of either AcSDKP (0.1 or 2 mg/kg) or saline on day 9. Captopril alone dose-dependently increased plasma AcSDKP by a factor of 3 to 5 and urine AcSDKP by a factor of 3. It slightly increased renal and pulmonary AcSDKP concentrations but did not affect AcSDKP concentrations in bone marrow and spleen. The combination of AcSDKP (2 mg/kg) and captopril gave very high AcSDKP concentrations in plasma and urine and increases in AcSDKP concentration by factors of 27 in kidney, 5.5 in lung, and 6.9 in the extracellular fraction of bone marrow. In contrast, no change was observed in the AcSDKP concentration in spleen and in the intracellular fraction of bone marrow. In conclusion, during chronic ACE inhibition in rats, AcSDKP levels slightly increased in organs with high ACE contents. No such increase occurred in hematopoietic organs. AcSDKP had to be combined with captopril to significantly increase its concentration in tissues other than the spleen. The possibility of pharmacologically increasing AcSDKP levels in the extracellular fraction of bone marrow may be of value for protecting hematopoietic cells from the toxicity of cancer chemotherapy.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1998

The hemoregulatory peptide N-acetyl-ser-asp-lys-pro impairs angiotensin I-induced contractions in rat aorta.

The hemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is degraded by ACE. This study was designed to examine the effect of Ac-SDKP on the contractions to angiotensin I. Experiments were performed on rat aortic rings with endothelium exposed to nitro-L-arginine. Ac-SDKP (10 and 100 microM) significantly augmented angiotensin I ED20 (from 2.0+/-0.4 to 4.2+/-1.0 and 5.0+/-0.9 nM) and ED50 (from 4.3+/-0.7 to 8.6+/-1.0 and 10.7+/-1.3 nM, respectively), but did not alter its maximal response. The contractions to angiotensin II were not affected by Ac-SDKP. No degradation of exogenous Ac-SDKP nor detectable release of endogenous Ac-SDKP were observed in the incubation medium. These results suggest that Ac-SDKP impairs angiotensin I response by inhibiting ACE and subsequent angiotensin II formation.

Angiotensin I↗

PubMed1998

Bromocriptine modulates P-glycoprotein function.

The multidrug resistance (MDR)-associated P-glycoprotein (P-gp) is a membrane transporter which carries, at the expense of MgATP hydrolysis, many amphiphilic molecules, such as the MDR-related cytotoxic drugs vincristine and vinblastine, and the MDR-reversing agents verapamil and progesterone. We have tested the effects on P-gp function of bromocriptine (BCT), an ergot alkaloid known as a D2 dopaminergic receptor agonist. BCT (at 4 microM) partially reverses the P-gp-mediated vincristine resistance of the Chinese hamster lung fibroblasts DC-3F/ADX, a MDR cell line. P-gp containing membrane vesicles prepared from the DC-3F/ADX cells exhibit, in the absence of any added drug, a basal MgATPase activity due to P-gp. BCT inhibits this basal ATPase activity, with a half-inhibiting concentration of 0.30 +/- 0.15 microM. BCT also inhibits the verapamil-induced P-gp ATPase stimulation competitively (Ki approximately 0.2 microM), and the progesterone-induced P-gp ATPase stimulation non-competitively (Ki approximately 0.07-0.10 microM). BCT also non-competitively inhibits the vinblastine-dependent P-gp ATPase activity within the same concentration range. Hydroxylated metabolites of BCT have different effects on P-gp ATPase, only the monohydroxylated being able to modulate both the basal and the drug-stimulated ATPase activities. In conclusion, these effects of BCT on P-gp function can be linked to a specific interaction with P-gp, probably involving inhibition of P-gp-mediated drug transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Lisinopril, an angiotensin I-converting enzyme inhibitor, prevents entry of murine hematopoietic stem cells into the cell cycle after irradiation in vivo.

The hemoregulatory peptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been shown in vivo to inhibit the cycling of murine hematopoietic stem cells triggered into S-phase by either cytotoxic drug administration or irradiation. This property, further confirmed using in vitro models, demonstrates that the peptide has an in vivo protective effect on the hematopoietic system. AcSDKP has been shown to be a physiological substrate of angiotensin I-converting enzyme (ACE), which catabolizes the peptide through a dipeptidasic activity. Thus, oral administration of ACE inhibitor to humans has led to an increase in the plasma AcSDKP concentration. In the present paper, we report on the in vivo effect of lisinopril, an ACE inhibitor, on the proliferative status of murine hematopoietic stem cells triggered into S-phase by irradiation. Administration of lisinopril (10 mg/kg) 1 hour after irradiation led to a 90 to 100% inhibition of murine plasma ACE activity as observed during the first 4 hours postirradiation. This inhibition was correlated with a 600% increase in the endogenous plasma AcSDKP level and a total suppression at 24 hours of entry of the hematopoietic stem cell into the cell cycle. We discuss the possible role of ACE in the regulation of hematopoietic stem cell proliferation through control of the AcSDKP concentration.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1997

A competitive enzyme hybridization assay for plasma determination of phosphodiester and phosphorothioate antisense oligonucleotides.

An enzyme competitive hybridization assay was developed and validated for determination of mouse plasma concentrations of a 15mer antisense phosphodiester oligodeoxyribonucleotide and of two phosphorothioate analogs. Assays were performed in 96-well microtiter plates. The phosphodiester sense sequence was covalently bound to the microwells. The 5'-biotinylated antisense sequence was used as tracer. The principle of the assay involves competitive hybridization of tracer and antisense nucleotide to the solid phase-immobilized sense oligonucleotide. Solid phase- bound tracer oligonucleotide was assayed after reaction with a streptavidin-acetylcholinesterase conjugate, using the colorimetric method of Ellman. As in competitive enzyme immunoassays, coloration was inversely related to the amount of analyte initially present in the sample. The limit of quantification was 900 pM for phosphodiester antisense oligonucleotide using a 100 microl volume of plasma without extraction. Cross-reactivity was negligible after a four base deletion in either the 3'or 5'position. The assay was simple and sensitive, suitable for in vitro screening of oligonucleotide hybridization potency in biological fluids and for measuring the plasma pharmacokinetics of phosphorothioate and phosphodiester sequences.

Animals↗

PubMed1997

An enzyme immunoassay for rat growth hormone: validation and application to the determination of plasma levels and in vitro release.

A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r = 0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n = 24) and 14% (n = 9) respectively. Minimal detectable amounts of rGH were 0.6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.

Acetylcholinesterase↗

PubMed1997

Effect of variability of plasma interferences on the accuracy of drug immunoassays.

Most immunoassays applied to drugs in human plasma do not use an extraction of analyte. To compensate for interferences due to plasma proteins or salts, standards are prepared in drug-free plasma. Because the concentration of plasma components varies from one subject to another, it is likely that the drug-free plasma is not representative of the potential interference in each plasma. Using two immunoassays, for a steroid (nomegestrol acetate) and a heptapeptide (BN 52080), the authors have shown that tracer binding to the antibody may vary significantly between plasma from different subjects. Intersubject variability of tracer-antibody binding was 21.6% (coefficient of variation for 25 subjects) for nomegestrol acetate. When the same plasma were spiked with the steroid at a concentration corresponding to the central part of the standard curve, the recovery was between 39 and 215%. Intersubject variability in tracer binding was lower (7.7%) for the peptide immunoassay, but still affected accuracy. The authors show that this problem is common to direct immunoassays for other drugs and must be solved in assay development.

Drug Monitoring↗

PubMed1997

High plasma level of N-acetyl-seryl-aspartyl-lysyl-proline: a new marker of chronic angiotensin-converting enzyme inhibition.

The acute administration of the angiotensin-converting enzyme (ACE) inhibitor captopril to healthy subjects transiently increases 5.5-fold the plasma levels of a natural stem-cell regulator, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). The aim of this study was to measure plasma Ac-SDKP levels during chronic treatment with all types of ACE inhibitors and to assess its relevance as a marker of ACE inhibition. Plasma levels of Ac-SDKP were blindly determined in age- and sex-matched hypertensive patients either treated (ACEI group, n=27) or not (non-ACEI group, n=23) with an ACE inhibitor for more than 1 month. Geometric mean [range] of plasma Ac-SDKP levels were significantly higher in the ACEI group (3.78 [1.48 to 14.5] pmol/mL) than in the non-ACEI group, with no overlap between the groups (0.75 [0.36 to 1.22] pmol/mL, P<.0001). The measurement of Ac-SDKP in plasma discriminated all the patients of the ACEI group, whereas the simultaneous determination of either in vitro (using hippuryl-histidine-leucine as substrate) or in vivo (angiotensin II/angiotensin I ratio) ACE activity failed to identify nine and five cases, respectively. We conclude that Ac-SDKP accumulates in plasma during chronic ACE inhibitor treatment. The long-term consequences of Ac-SDKP accumulation are unknown. The reliability of plasma Ac-SDKP measurement makes it the best marker of chronic ACE inhibition, which can help to verify patients' compliance to ACE inhibitor treatment.

Adolescent↗

PubMed1997

Metabolite involvement in bromocriptine-induced prolactin inhibition in rats.

Bromocriptine (BCT) is a dopamine D2 receptor agonist used for the treatment of Parkinson's disease and hyperprolactinemic disorders. After oral administration, BCT is metabolized into mono- or dihydroxylated metabolites. To study how these metabolites influence parent drug pharmacodynamics, we administered BCT to rats intravenously (1 mg/kg i.v.) and orally (10 mg/kg p.o.) and measured the inhibition of prolactin secretion. Despite similar areas under the curve for BCT, the duration of the effect was 36 h after oral and only 18 h after intravenous administration. Pharmacokinetic/pharmacodynamic models were used to correlate the concentration of BCT in the effect compartment with the lowering of prolactin. One of these models (effect compartment model) showed that the effective concentration (EC50) at the site of action was much lower after oral (0.56 nM) than after intravenous administration (3.68 nM). In contrast, the EC50 values based on BCT metabolite data were in the same range for both administrations. These observations suggested the activity of one or more BCT metabolites. To confirm this hypothesis, hydroxylated metabolites of BCT (produced in vitro by rat liver microsomes) were administered i.v. (100 microg/kg) in rats. We found that monohydroxylated BCT was able to lower prolactin secretion like BCT. Dihydroxylated metabolites, as well as monohydroxylated metabolites, were effective in reducing in vitro prolactin secretion. Because we demonstrated that the concentration of hydroxylated metabolites after oral administration is 55-fold that of BCT, it can be concluded that BCT activity in the pituitary after oral administration is mediated by its metabolites.

Animals↗

PubMed1996

Acute angiotensin-converting enzyme inhibition increases the plasma level of the natural stem cell regulator N-acetyl-seryl-aspartyl-lysyl-proline.

Angiotensin I-converting enzyme (ACE) has two homologous active NH2- and COOH-terminal domains and displays activity toward a broad range of substrates. The tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has been shown to be hydrolyzed in vitro by ACE and to be a preferential substrate for its NH2-terminal active site. This peptide is a regulatory factor of hematopoiesis which reversibly stem cells and normal early progenitors into S-phase. We found that a single oral dose of 50 mg of the ACE inhibitor, captopril, when administered to eight healthy subjects in a double-blind, crossover, placebo-controlled study, massively increased the plasma level of Ac-SDKP. ACE inhibition by captopril induced a 90-99% inhibition of in vitro [3H]Ac-SDKP hydrolysis and a long-lasting 5.5-fold (range: 4-8.5-fold) increase in the plasma levels of Ac-SDKP. These results demonstrate that Ac-SDKP is the first natural peptide hydrolyzed by the NH2-terminal domain of ACE not only in vitro but also in vivo, confirming that both catalytic sites of ACE are physiologically active. Our data suggest that ACE may also be implicated in the process of hematopoietic stem cell regulation, by permanently degrading this natural circulating inhibitor of cell entry into S-phase.

Administration, Oral↗

PubMed1996

Enzyme immunoassays for bromocriptine and its metabolites.

We have developed two bromocriptine enzyme immunoassays with different specificities for applications in human and animal pharmacokinetic studies. The first assay uses antibodies directed against the cyclopeptide structure of bromocriptine, and is specific for untransformed bromocriptine. The second assay uses antibodies directed against the bromolysergic part of the molecule and allows the measurement of both bromocriptine and its metabolites. Enzymatic tracers were obtained by covalent coupling of bromocriptine analogs to acetylcholinesterase from the electric eel Electrophorus electricus. Both assays have a limit of detection of 10 pg/ml and a limit of quantification of 50 pg/ml. The specificity of the assays was determined following fractionation by high-performance liquid chromatography of rat samples obtained after administration of bromocriptine.

Administration, Oral↗

PubMed1996

Bioequivalence study of alpha-dihydroergocryptine: utility of metabolite evaluation.

Metabolite assessment is an open question in bioequivalence studies. In situations of low absorption, high first-pass metabolism, and intrasubject variability, metabolites may reflect absorption more adequately than the parent drug, and their determination may help decision-making in bioequivalence issues. Treating alpha-dihydroergocryptine (DHECT) as a model, we used both unchanged DHECT and a pool of DHECT metabolites to evaluate the bioequivalence of 2 oral DHECT formulations (reference-R and test-T) in 12 subjects. DHECT and its metabolites were immunoassayed. There was no difference between the 2 formulations in terms of the AUC0-infinity (area under the curve) values determined from unchanged DHECT or DHECT with metabolites profiles: 572 +/- 490 pg/ml.h (R) and 442 +/- 276 pg/ml.h (T) for unchanged DHECT, and 7,141 +/- 2,936 pg/ml.h (R) and 6,941 +/- 1,462 pg/ml.h (R) for DHECT with metabolites. Confidence intervals were within the ranges 0.8-1.25 (AUC0-infinity) and 0.7-1.43 (Cmax) for DHECT with metabolites but not for unchanged DHECT. This study describes a particular case where only measurements on the basis of the metabolites can justify the assumption of bioequivalence.

Adrenergic alpha-Antagonists↗

PubMed1996

Two different approaches for developing immunometric assays of haptens.

To improve immunoassays of small haptens, we developed two different approaches for their measurement in a non-competitive format. We first devised two-site immunometric assays for small peptides (8-11 amino acids) by selecting two sets of antibodies specifically directed against C- and N-terminal moieties of the peptides. In each case, assay sensitivity improved substantially over that of the corresponding competitive assays. More interestingly, all of these new immunometric assays were much more specific than the competitive assays. In a second approach, we developed a new procedure, solid-phase-immobilized epitope immunoassay (SPIE-IA), in which a single monoclonal antibody uses the same epitope for capture and tracer binding and the hapten is covalently cross-linked to solid-phase proteins. To date, SPIE-IA have been successfully applied to the determination of haptens bearing primary amino groups, including substance P, thyroxine, leukotriene C4, endothelin, and angiotensin II. In each case, assay sensitivity was significantly improved.

Amino Acid Sequence↗

PubMed1995

Immunometric assay of BN 52080, a heptapeptide C-terminal analogue of sorbin.

A novel type of enzyme immunometric assay has been developed for a heptapeptide, BN 52080. This compound is a short C-terminal analogue of sorbin and is under clinical evaluation for treatment of chronic diarrhea. In this solid-phase immobilized epitope immunoassay (SPIE-IA), the peptide is first immunologically bound to polyclonal antibodies adsorbed to a solid phase and then, after covalent immobilization with glutaraldehyde, is released from the antibody paratope by NaOH. The peptide linked to the solid phase is further quantified with a tracer consisting of the same antibodies purified by affinity chromatography and coupled to acetylcholinesterase. This assay has a detection limit of 10 pg/ml and is therefore five times more sensitive than competitive enzyme immunoassay using the same antibodies and BN 52080 coupled to acetylcholinesterase as tracer. The assay is specific and allows direct measurement of peptide in human plasma after subcutaneous or intravenous administration of 200 micrograms of BN 52080 to volunteers.

Amino Acid Sequence↗

PubMed1994

Enzyme immunometric assay for leukotriene C4.

An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.

Acetylcholinesterase↗

PubMed1994

Recombinant technology in the preparation of immunogen and enzymatic tracer. Application to the development of an enzyme immunoassay for rat prolactin.

A competitive enzyme immunoassay for rat prolactin using an immunogen and a tracer obtained by recombinant DNA technology is described. Polyclonal antibodies were raised in rabbits immunized with a purified chimeric protein consisting of rat prolactin fused with two synthetic immunoglobulin G binding domains derived from staphylococcal Protein A. The enzymatic tracer was obtained using an expression system which permits insertion of rPRL DNA sequence in the alkaline phosphatase gene. Antibodies and tracer were used to develop a solid-phase competitive immunoassay for the measurement of rat prolactin in plasma with a minimal detectable concentration of 0.5 ng/ml. The mean intra- and inter-assay coefficients of variation were 7.8 and 13.2%, respectively. Rat plasma concentrations measured with this assay correlated well with those obtained with a conventional enzyme immunoassay (r = 0.993, slope = 1.037, n = 24).

Alkaline Phosphatase↗

PubMed1994

Effect of chronic ANG I-converting enzyme inhibition on aging processes. I. Kidney structure and function.

The effect of angiotensin I-converting enzyme inhibition (ACEI) on the age-related changes in the kidney structure and function was investigated in rodents. Normotensive male Wistar (WAG)/Rij rats were treated with perindopril from the age of 6 mo to the day of killing at 12, 24, or 30 mo. Mean blood pressure, constant from 6 to 30 mo, was reduced by 19 mmHg in treated animals. With age, the major functional modifications were a decrease in glomerular filtration rate and in renal blood flow, a rise in intrarenal vascular resistance (IVR), a reduced tubular reabsorption of salts, and a progressive increase in proteinuria. ACEI significantly reduced IVR and proteinuria. From a structural point of view, the glomeruli showed 1) an increase in size, 2) a decrease in capillary surface, 3) a diffuse thickening of the glomerular basement membrane, 4) an expansion of the mesangial matrix, and 5) an accumulation of albumin droplets in podocytes inducing 6) a dispersed focal and segmental glomerulosclerosis which, at 30 mo, affected < 2% of glomeruli. Of these six age-related structural changes, ACEI delayed the appearance of the three latter changes.

Aging↗