Pone.0010914 1.9

Design of Group IIA Secreted/Synovial Phospholipase A2Inhibitors: An Oxadiazolone Derivative SuppressesChondrocyte Prostaglandin E2 Secretion Jean-Edouard Ombetta1., Natacha Thelier2., Chang Zhi Dong3, Ste´phanie Plocki3, Lydia Tsagris2, Franc¸ois Rannou2,4, France Massicot5, Atime´ Djimde´3, Elissar El-Hayek2, Yiming Shi3, Franc¸oise Heymans3, Nohad Gresh6, Caroline Chauvet2* 1 Laboratoire de Chimie Organique, Faculte´ de Pharmacie, Universite´ Franc¸ois Rabelais, Tours, France, 2 Laboratoire de Pharmacologie, Toxicologie et Signalisation Cellulaire, INSERM UMR-S-747, UFR Biome´dicale des Saints Pe res, Universite´ Paris Descartes, Paris, France, 3 Equipe de Pharmacochimie, ITODYS, CNRS UMR7086, Universite´ Paris Diderot, Paris, France, 4 Service de re´e´ducation, AP-HP, Hoˆpital Cochin, Paris, France, 5 Laboratoire de Chimie-Toxicologie analytique et cellulaire, EA4463, Faculte´ de Pharmacie, Universite´ Paris Descartes, Paris, France, 6 Laboratoire de Chimie et Biochimie Pharmacologique et Toxicologique, CNRS UMR8601, UFR Biome´dicale des Saints Pe res, Universite´ Paris Descartes, Paris, France Group IIA secreted/synovial phospholipase A2 (GIIAPLA2) is an enzyme involved in the synthesis of eicosanoids such asprostaglandin E2 (PGE2), the main eicosanoid contributing to pain and inflammation in rheumatic diseases. We designed, bymolecular modeling, 7 novel analogs of 3-{4-[5(indol-1-yl)pentoxy]benzyl}-4H-1,2,4-oxadiazol-5-one, denoted C1, aninhibitor of the GIIAPLA2 enzyme. We report the results of molecular dynamics studies of the complexes between thesederivatives and GIIAPLA2, along with their chemical synthesis and results from PLA2 inhibition tests. Modeling predictedsome derivatives to display greater GIIAPLA2 affinities than did C1, and such predictions were confirmed by in vitro PLA2enzymatic tests. Compound C8, endowed with the most favorable energy balance, was shown experimentally to be thestrongest GIIAPLA2 inhibitor. Moreover, it displayed an anti-inflammatory activity on rabbit articular chondrocytes, as shownby its capacity to inhibit IL-1b-stimulated PGE2 secretion in these cells. Interestingly, it did not modify the COX-1 to COX-2ratio. C8 is therefore a potential candidate for anti-inflammatory therapy in joints.
Citation: Ombetta J-E, Thelier N, Dong CZ, Plocki S, Tsagris L, et al. (2010) Design of Group IIA Secreted/Synovial Phospholipase A2 Inhibitors: An OxadiazoloneDerivative Suppresses Chondrocyte Prostaglandin E2 Secretion. PLoS ONE 5(6): e10914. doi:10.1371/journal.pone.0010914 Editor: Sudha Agarwal, Ohio State University, United States of America Received January 19, 2010; Accepted April 1, 2010; Published June 1, 2010 Copyright: ß 2010 Ombetta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported in part by INSERM, University Paris Descartes, Arthritis Foundation Courtin and a program of Paris Centre University (ParisDescartes and Paris Diderot). The authors thank the computer center CINES-Montpellier for computer time and technical support. The authors also thank theLigue Nationale Contre le Cancer for support. NT was supported by Assistance Publique-Hopitaux de Paris. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: caroline.chauvet@parisdescartes.fr . These authors contributed equally to this work.
ever, COX-2 is absent in most tissues under normal restingconditions but is induced in inflamed tissues and is responsible for Inflammation is a multi-faceted process involving numerous increased PGE2 production. This activation has motivated the enzymes, such as phospholipases A2 (PLA2s) and cyclo-oxygenases development of selective COX-2 inhibitors. However, these (COXs) [1]. PLA2s catalyze the hydrolysis of cell-membrane inhibitors also have severe side effects such as myocardial glycerophospholipids at the sn-2 position leading to the generation infarction [5,6]. Overcoming this problem could involve the of free fatty acids such as arachidonic acid. The later is development of novel anti-inflammatory agents to efficiently subsequently metabolized into potent pro-inflammatory mediators inhibit the PLA2-dependent production of COX substrates such as eicosanoids (e.g. prostaglandin E2 [PGE2]) through a without impairing the balance between COX-1 and COX-2.
pathway involving COX-1 and COX-2 in part [2]. PGE2 is the PLA2s represent a growing family of enzymes of two main main eicosanoid contributing to pain and inflammation in categories, intracellular and secreted. Among the 10 human rheumatic diseases [3,4]. Nonsteroidal anti-inflammatory drugs secreted PLA2s (sPLA2s) known to date, the most studied is the (NSAIDs) reduce the production of PGE2, which leads to a non-pancreatic Group IIA, denoted GIIAPLA2, because of its significant improvement in rheumatic symptoms. However, these involvement in the pathogenesis of many inflammatory diseases drugs exhibit gastrointestinal toxicity mainly because of a marked (for a review, see [7]). GIIAPLA2 was originally purified from the decrease in COX-1 activity [4] and renal and blood pressure synovial fluid of patients with rheumatoid arthritis [8,9,10]. The toxicities mainly because of a decrease in COX-2 activity. COX-1 number of rheumatoid arthritis-affected joints and the presence of is constitutively expressed in most tissues and appears to be destructive erosion correlate with the amount of GIIAPLA2 in the responsible for maintaining normal physiological function. How- serum of patients [11]. Moreover, GIIAPLA2 induces an PLoS ONE www.plosone.org June 2010 Volume 5 Issue 6 e10914 PLA2 Inhibitors in Chondrocyte inflammatory response when injected in rabbit joints [12] and inhibitory potencies of C2 to C8 against GIIAPLA2 was analyzed exacerbates rat adjuvant arthitis after intradermal injection [13].
by enzymatic assay, and the anti-inflammatory activity of the most The systemic implication of sPLA2s in inflammation has potent compound, C8, was evaluated in IL-1b-treated articular prompted a number of research groups to develop selective inhibitors of different types of these enzymes. Some potentcandidates have been evaluated in phase II clinical trials.
Surprisingly, no effect was observed when such inhibitors wereused to treat patients with sepsis or rheumatoid arthritis [14,15].
Molecular modeling This failure could be due to the complexity of the inflammation We previously reported that one of the essential interactions process and the existence of compensatory pathways. However, between C1 and the target GIIAPLA2 is Ca(II) bidentate chelation these molecules have been tested only in high-level systemic by the oxadiazolone moiety in its anionic form [20]. Because C2- inflammatory diseases, not in low-level inflammatory diseases such C8 are structurally similar to C1 (Fig. 1), docking was performed as atherosclerosis, diabetes, Alzheimer's, and osteoarthritis.
upon first anchoring the oxadiazolone ring in the same position as Varespladib, a sPLA compound C1, followed by energy minimization and molecular 2 inhibitor, was recently found to reduce atherosclerosis in apolipoprotein-E-null mice [16]. Thus, the dynamics. As was observed for compound C1, the lowest-energy frames of C2-C8/enzyme complexes are stabilized by p–p and inhibitors in these low-level inflammatory diseases should be re-examined.
cation-p interactions involving His6, Phe23, and Phe63 on the one We have developed various selective inhibitors of sPLA hand and Arg7 and Arg33 of GIIAPLA 2 on the other. Table 1 lists [17,18,19,20,21]. Previously, we reported on the computer- the energy values corresponding to the lowest-energy frames from assisted design and synthesis of a series of novel oxadiazolone molecular dynamics.
derivatives that were shown to exhibit potent inhibitory propertiesagainst GIIAPLA 2 [20]. In this series, a Ca(II)-binding oxadiazo- lone ring was connected through a polymethylene chain of varying As outlined in Figure 1, 4-(5-bromopent-1-yloxy)benzyl cyanide lengths to an indole ring, which has been shown to be involved in 1 is prepared according to Dehaen and Hassner [22] by mono- apolar and cation-p interactions with GIIAPLA2 residues. The substitution of 1,5-dibromopentane with 4-hydroxybenzyl cyanide optimal length of the linker was found to encompass 5 methylenes, in moderate yield. Compound 1 is then condensed in 25% to 50% and the corresponding compound, (3-{4-[5-(indol-1-yl)pentoxy]- yields, with 5-substituted indole derivatives or different aromatic benzyl}-4H-1,2,4-oxadiazol-5-one), is denoted C1 in the present alcohols, through their sodium salts prepared prior to use, to give study. In the current work, the indole moiety was replaced by 2a–g. The nitrile function of 2a–g is converted into amidoxime, other aromatic groups, which gave rise to compounds C2 to C8.
by use of hydroxylamine released in situ from its HCl salt, to Using molecular modeling, we computed and ranked energy provide 3a–g in 35% to 80% yields. The action of phenyl balances for the binding of these inhibitors to GIIAPLA2. The chloroformate to the amidoximes 3a–g leads to the corresponding Figure 1. Synthesis scheme. Reagents and conditions: (a) Br(CH2)5Br, K2CO3, DMF, RT, 10 days; (b) Ar-NH, K2CO3, CH3CN, reflux; (c) NaOH, AbsEtOH; (d) 1, DMF, RT, 24 h; (e) NH2OH-HCl, K2CO3, Abs EtOH; (f) PhOCOCl, Et3N, CH2Cl2; (g) toluene, reflux. The terms 1, 2a-g and 3a-g written in boldrefer to the C2-C8 precursors. The terms a to g written in bold in the bottom of the figure refer to the radicals (R) of the C2 to C8 compounds,respectively. The radical of the C1 compound is also shown.
doi:10.1371/journal.pone.0010914.g001 PLoS ONE www.plosone.org June 2010 Volume 5 Issue 6 e10914 PLA2 Inhibitors in Chondrocyte Rekker's fragmental data [24] (Table 2). The molecules C1-C8 Table 1. Energy balances (e = 4) from performing single-point are specific inhibitors of hGIIAPLA2 because none inhibited Poisson-Boltzmann calculations of continuum solvation pGIBPLA2 at the highest concentration tested (100 mM). Such selectivity implies that C1-C8 should not interfere with thedigestion process.
The experimentally measured IC 50s for hGIIAPLA2 (Table 2) Esolvlig Esolvprot dEsolv dE2 are associated with the final energy balances, denoted dE2 in 2115.9 2344.3 222.5 2385.1 Table 1. The ranking of C1-C8 in terms of IC50 is the same as 2113.4 2343.6 223.9 2385.1 that of the dE2 magnitudes. In C2, the second phenyl ring is 2114.9 2344.1 222.7 2385.1 substituted with the ether O in the ortho position and in C6 in thepara position. Both IC 2116.5 2343.7 221.7 2385.1 50 and dE2 values show C2 to have a significantly enhanced affinity for PLA2 as compared with C6, 2116.2 2350.0 221.8 2385.1 even though both are iso-lipophilic (Tables 1 and 2). In C2, the 2111.3 2350.1 221.7 2385.1 biphenyl group has favorable van der Waals interactions with both 2109.0 2340.2 221.8 2385.1 Phe23 and Val30 of the enzyme, but in C6, the interactions are 2118.2 2349.7 221.8 2385.1 limited to Phe23. Such interactions could be further optimized, aswhen the biphenyl ring was replaced by phenantrene in C8. The All energies are given in kcal/mol. Eint denotes the inhibitor (compounds C1 to lowest-energy complex is now stabilized by an enhanced overlap of C8)-protein interaction energy, and dElig and dEprot the costs of conformational this ring with Phe63 (Fig. 2). However, the lipophilicity increases in energy rearrangements of the inhibitor and the protein, respectively, onpassing from their free to complexed states, and dE parallel, which could possibly limit the bioavailability of C8. We 1 is the sum: Eint+ dElig + dEprot. dE1 corresponds to a gas-phase complexation energy. Esolvprot and Esolvlig found C8 indeed endowed with the most favorable dE2 value denote the continuum solvation energies of the isolated protein and the ligand, (Table 1), which was experimentally associated with the lowest respectively, following gas-phase energy minimization in the absence of IC50 value (0.62 mM vs. 5 mM for C1).
complexation. They represent the energy cost necessary to dehydrate bothentities prior to complex formation. E At the other extreme, replacing the C1 indole ring by the solvtot denotes the continuum solvation energy of the complex. Thus, dEsolv = Esolvtot – Esolvlig – Esolvprot, which smaller and less electron-rich phenyl ring, as in C7, resulted in a represents the resulting solvation energy balance. The overall energy balance reduction of 10.3 kcal/mol in dE2 value. Thus, C7 can be including solvation is denoted as dE2 = dE1 + dEsolv.
predicted to have the least inhibitory potency in the series. This finding was confirmed by experimentation showing C7 to have thehighest IC carbonate intermediates, which, when heated to reflux of toluene, 50 value (35 mM vs. 5 mM for C1).
Similar to C1, compounds C3-C5 have a bicyclic ring, whereas cyclizes intra-molecularly to generate the substituted oxadiazo- C3 possesses a benzo-1,3-thiazole instead of an indole ring. C4 lones C2-C8 in 34% to 50% yields.
and C5 have a chlorine and a methoxy substituent, respectively, inposition 5 of the indole. In C3-C5, the aromatic rings interact In vitro inhibition of enzymatic activity of sPLA2s by C1- simultaneously with His6, Arg7, and Val3, as was previously observed for C1 [20]. The difference in activity between C4 and The compounds C1-C8 were submitted to fluorimetric assay to C5 could be explained by additional electrostatic and/or van der determine their inhibitory potencies and selectivity towards human Waals interactions contributed by methoxy substitution. C3 has GIIAPLA2 (hGIIAPLA2) versus porcine group IB PLA2 (pGIB- anti-hGIIAPLA2 activity close to that of C1, along with PLA2) (Table 2). GIBPLA2 is an enzyme of the same family as substantially reduced lipophilicity (2.88 vs. 3.81 for C1).
GIIAPLA2 (sPLA2) but is mainly involved in digestion of dietary Thus, in the C1-C8 series, C8 has the most favorable dE2 value phospholipids and is secreted by the pancreas [23]. Lipophilicity and the lowest IC50 on human GIIAPLA2 activity, as evaluated by parameters, log P, of these products are calculated by use of enzymatic assay. On the bases of the IC50 values we focused ourcellular assays on the most potent compound C8, the solecompound with a sub-micromolar activity. We thus chose to Table 2. Inhibition of enzymatic activities of porcine evaluate the cytotoxicity and anti-inflammatory activity of C8 in pancreatic group IB (pGIB) and human group IIA (hGIIA) PLA2s primary cultured rabbit articular chondrocytes treated with the by compounds C1 to C8 and their corresponding log P pro-inflammatory cytokine IL-1b, which is known to play a key role in rheumatic diseases such as osteoarthritis (for reviews see[25,26]). Chondrocyte is the unique cell type in joints, and the cellmodel we chose is widely used to study the effect of inflammatory stress on joint cells.
Evaluation of the cytotoxicity of C8 on articular We assessed the viability of the chondrocytes by MTT assay to evaluate the cytotoxic effects of C8 on these cells. Chondrocytes were treated for 20 h with 1 ng/mL IL-1b alone or 1 h after the addition of C8 at 0.31 to 9.92 mM, which corresponded to 0.5- to 5-fold the IC50 of C8 on human GIIAPLA2 activity (Table 2).
Three different culture medium compositions were used: DMEM alone, or supplemented with 0.1% BSA or 2% FCS. IL-1b had no cytotoxic effects as compared with the untreated control condition *: calculated using the Rekker's hydrophobic fragmental constants for the three culture media tested (Fig. 3). In chondrocytes cultured in DMEM alone but with IL-1b, C8 had no cytotoxic effects at PLoS ONE www.plosone.org June 2010 Volume 5 Issue 6 e10914


PLA2 Inhibitors in Chondrocyte Figure 2. Representation of the most important interactions between C8 and the binding site of hGIIAPLA2 found from modeling.
The structure presented in the figure was derived from molecular dynamics using the Accelrys software and the cff91 force field (see SupportingInformation S1). In this presentation are shown in particular: a) the chelating of Ca(II) by the oxadiazolone moiety of C8, as well as Gly29 (G29), Gly31(G31) and Asp48 (D48) of hGIIAPLA2 with their main-chain or side-chain carbonyls; b) the phenoxy binding site [(Leu2 (L2), Phe5 (F5), His6 (H6) andAla18 (A18)]; and c) the binding site of 5-(phenanthren-9-yloxy)pentyl [Asn1 (N1), Val3 (V3) and Phe63 (F63)].
doi:10.1371/journal.pone.0010914.g002 0.31 to 2.48 mM (Fig. 3A). In chondrocytes cultured in DMEM concentrations of 0.31-, 0.62-, 0.94-, and 1.24-mM, C8 decreased with 0.1% BSA or 2% FCS and IL-1b, C8 had no cytotoxic effects the production of PGE2 induced by IL-1b by 59-, 58-, 74-, and 80-%, at 0.31 to 9.92 mM (Fig. 3B and 3C). Thus, we evaluated the anti- respectively (Fig. 4A). In DMEM supplemented with 0.1% BSA, at inflammatory activity of C8 in culture conditions from 0.31 to concentrations of 0.31-, 0.62-, 0.94-, 1.24-, 2.48-, and 4.96-mM, C8 1.24 mM in DMEM alone and from 0.31 to 4.96 mM in DMEM decreased the production of PGE2 induced by IL-1b by 31-, 30-, 45-, supplemented with 0.1% BSA or 2% FCS.
43-, 81-, and 92-%, respectively (Fig. 4B). In DMEM supplementedwith 2% FCS, at concentrations of 0.31-, 0.62-, 0.94-, 1.24-, 2.48-, Effect of C8 on IL-1b-stimulated PGE2 secretion in and 4.96-mM, C8 decreased the production of PGE2 induced by IL- articular chondrocytes 1b by 26-, 48-, 49-, 54-, 68-, and 68-%, respectively (Fig. 4C). It is We tested the effect of C8 on the IL-1b-stimulated secretion of important to note that C8 down-regulated the IL-1b-stimulated 2 in chondrocytes. PGE2 synthesis takes place mainly in response 2 to the level of the control untreated condition at to cell activation by IL-1b, and its generation accounts for many of 4.96 mM in DMEM supplemented with 0.1% BSA and at 2.48 and the actions induced by this cytokine [27]. In vitro, IL-1b induces the 4.96 mM in DMEM supplemented with 2% FCS. The effect of C8 expression of COX-2 by chondrocytes, which results in increased was then evaluated at the extreme concentrations (0.31- and 4.96- mM) in DMEM supplemented with 2% FCS and containing 2 production [28]. PGE2 release thus represents a powerful IL- decreasing (1-, 0.5-, and 0.25-ng/mL) IL-1b concentrations 2-dependent inflammatory marker in our cell model.
Chondrocytes were treated for 20 h with IL-1b alone or 1 h after the (Table 3). The anti-IL-1b inhibitory effect of C8 at 0.31 mM addition of C8. As expected, IL-1b significantly stimulated PGE increases when IL-1b concentration decreases. The inhibitory effect secretion by chondrocytes in the three different culture media: 23.3-, of C8 at 4.96 mM does not change when IL-1b concentration 18.3- and 2.8-fold induction as compared with untreated control decreases. This is probably due to the fact that at 4.96 mM, the conditions, in DMEM alone or supplemented with 0.1% BSA or 2% inhibitory effect of C8 on IL-1b-induced PGE2 production is FCS, respectively (Fig. 4). In chondrocytes treated with IL-1b, C8 maximal. A parallel cellular test was performed on the compound C1 had a strong and statistically significant inhibitory effect on PGE2 whose IC50 is 5 mM (Table 2) and we observed that a 8 mM dose of secretion at all concentrations tested: from 0.31 to 1.24 mM in C1, corresponding to 1.6-fold the IC50 of C1 on human GIIAPLA2 DMEM alone or from 0.31 to 4.96 mM in DMEM supplemented activity, does not decrease the stimulated PGE2 secretion by IL-1b at with 0.1% BSA or 2% FCS (Fig. 4). In DMEM alone, at 1 ng/mL (data not shown). Thus, C8, but not C1, decreases the IL- PLoS ONE www.plosone.org June 2010 Volume 5 Issue 6 e10914



PLA2 Inhibitors in Chondrocyte Figure 4. Effect of IL-1b and C8 on PGE Figure 3. Effect of IL-1b and C8 on viability of articular 2 secretion by articular chondrocytes. Chondrocytes were untreated (white bars) or treated chondrocytes. Chondrocytes were untreated (white bars) or treated for 20 h with IL-1b alone (black bars) or 1 h after the addition of C8 for 20 h with IL-1b alone (black bars) or 1 h after the addition of C8 (grey bars) in DMEM alone (A) or with 0.1% BSA (B) or with 2% FCS (C).
(grey bars) in DMEM alone (A) or supplemented with 0.1% BSA (B) or 2% FCS (C). Cell viability was evaluated by an MTT-based test. Data 2 concentration was determined in conditioned culture medium, and protein concentration was determined in whole-cell protein represent the absorbance570nm - absorbance690nm and are expressed as extracts. Data represent the ratio of PGE relative arbitrary units, where the IL-1b-untreated group represents 2 concentration relative to whole cell protein concentration (pg PGE 100%. Values are means 6 SEM (n = 3 to 8 independent determina- 2/mg proteins). Values are means 6 SEM (n = 3 to 7 independent determinations). { P,0.05, tions). { P,0.05, {{ P,0.01, {{{ P,0.001 between untreated and IL-1b- {{ P,0.01, {{{ P,0.001 between untreated and IL-1b-treated groups; treated groups; * P,0.05, ** P,0.01, *** P,0.001 between IL-1b- and * P,0.05, ** P,0.01, *** P,0.001 between IL-1b- and IL-1b+C8-treated 1b-stimulated PGE2 secretion in a dose-dependent manner in the inflammatory responses. In vitro, IL-1b induces the expression of three culture medium compositions used.
inducible NO synthase (iNOS) by chondrocytes, and consequentlyan increase in NO production [29]. NO secretion, evaluated by Effect of C8 on IL-1b-stimulated NO secretion in articular nitrite concentration in the cell culture medium, represents a reliable IL-1b-dependent and PLA2-independent inflammatory We tested the effect of C8 on the IL-1b-stimulated secretion of marker in our cell model. Chondrocytes were treated for 20 h with NO in chondrocytes. NO is a mediator of immune and IL-1b alone or 1 h after the addition of C8. As expected, IL-1b PLoS ONE www.plosone.org June 2010 Volume 5 Issue 6 e10914


PLA2 Inhibitors in Chondrocyte Table 3. Effect of C8 on PGE2 secretion by articularchondrocytes incubated with different IL-1b concentrations.
C8 inhibitory effect (%inhibition) Chondrocytes were untreated or treated for 20 h with IL-1b (1-, 0.5-, or 0.25-ng/mL) alone or 1 h after the addition of C8 (0.31- or 4.96-mM) in DMEM with 2%FCS. PGE2 concentration was determined in conditioned culture medium, andprotein concentration was determined in whole-cell protein extracts. The PGE2concentration was normalized relatively to whole cell protein concentration (pgPGE2/mg proteins). The means of PGE2 concentrations from 3 independentdeterminations were calculated and the anti-IL-1b inhibitory effect of C8 wasdetermined with the formula %inhibition = 10021006[(mean PGE2 in IL-1b+C8condition)/(mean PGE2 in IL-1b condition)]).
*P,0.05,***P,0.001 between IL-1b- and IL-1b+C8-treated groups.
doi:10.1371/journal.pone.0010914.t003 significantly stimulated nitrite secretion by chondrocytes in thethree different culture media: 8.1-, 2.6-, and 2.0-fold induction ascompared with the control conditions, in DMEM medium aloneor supplemented with 0.1% BSA or 2% FCS, respectively (Fig. 5).
C8 did not significantly inhibit the IL-1b-stimulated nitritesecretion in chondrocytes cultured in DMEM medium alone orsupplemented with 0.1% BSA (Fig. 5A, B). In DMEMsupplemented with 2% FCS, C8 did not inhibit the IL-1b-stimulated nitrite secretion at 0.31-, 0.62-, and 0.94-mM andslightly decreased by 17-, 19-, 21-% the IL-1b-induced nitriteproduction at 1.24-, 2.48-, and 4.96-mM, respectively (Fig. 5C).
Thus, C8 did not inhibit the IL-1b-stimulated NO secretion inDMEM alone or supplemented with 0.1% BSA and slightlyinhibited IL-1b-stimulated NO secretion in DMEM supplementedwith 2% FCS.
Effect of C8 on COX-1, COX-2, and iNOS protein levels inarticular chondrocytes We evaluated the effect of C8 on COX-1, COX-2 and iNOS protein levels in chondrocytes treated with IL-1b. Chondrocyteswere treated for 20 h with IL-1b alone or 1 h after the addition ofC8 (0.31-1.24 mM) in DMEM, and protein extracts wereexamined by western blot analysis. As expected, COX-1 protein Figure 5. Effect of IL-1b and C8 on nitrite secretion by articular was detectable but COX-2 and iNOS proteins were undetectable chondrocytes. Chondrocytes were untreated (white bars) or treated in untreated control conditions (Fig. 6A). Moreover, IL-1b for 20 h with IL-1b alone (black bars) or 1 h after the addition of C8 treatment induced the expression of COX-2 and iNOS proteins (grey bars) in DMEM alone (A) or supplemented with 0.1% BSA (B) or 2% without affecting the level of COX-1 protein (Fig. 6A). In the FCS (C). NO secretion was indirectly evaluated by determination of presence of IL-1b, C8 did not alter the COX-1, COX-2 and nitrite concentration in conditioned culture medium. Protein concen-tration was determined in whole-cell protein extracts. Data represent iNOS protein levels (Fig. 6A). Consequently, the protein ratio of the ratio of nitrite concentrations relative to whole-cell protein COX-1 to COX-2 was not modified by C8 (Fig. 6B).
concentration (pmol nitrites/mg protein). Values are means 6 SEM(n = 4 to 7 independent determinations). { P,0.05, {{ P,0.01, {{{ P,0.001 between untreated and IL-1b-treated groups; * P,0.05,** P,0.01, *** P,0.001 between IL-1b- and IL-1b+C8-treated groups.
Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit COX-1 and COX-2, and selective COX-2 inhibitors are currentlyused to reduce rheumatic symptoms. However, these drugs exhibit ranked energy balances for the binding of these inhibitors to gastrointestinal, renal, blood pressure and cardiovascular toxici- GIIAPLA2. The energy balances (Table 1) taking into account ties. To overcome this problem, GIIAPLA2 inhibitors could be solvation effects show a correlation between dE2, the overall developed to inhibit the production of COX substrates without energy balance for binding, and the experimentally measured IC50 impairing the balance between COX-1 and COX-2. We designed for our novel compounds C1-C8. This finding should lend and synthesized 7 new oxadiazolone derivatives (C2 to C8) additional credence to our previous results [20], despite the derived from C1. Using molecular modeling, we computed and approximations of the computational approach used in that study, PLoS ONE www.plosone.org June 2010 Volume 5 Issue 6 e10914


PLA2 Inhibitors in Chondrocyte passing from compound C1 with an indole ring to C3 with abenzothiazole. Nevertheless, the high lipophilicity of C8 should bean interesting option for its prospective clinical development,considering the possibility of local administration (intra-articularinfiltration).
The toxicity and anti-inflammatory activity of C8 were evaluated in rabbit articular chondrocytes in primary culture.
The toxicity of C8 was assessed by MTT, which allows anevaluation of the cell number and/or metabolic activity in cells.
C8 (from 0.31 to 9.92 mM) did not decrease cell viability in culturemedium supplemented with 0.1% BSA or 2% FCS but did (at 4.96and 9.92 mM) in culture medium alone. This observation isprobably due to the cells being weakened in the absence of BSA orFCS. We also observed, as expected, an increase in cell numberand/or metabolic activity in response to IL-1b. This effectincreases in the presence of C8, at non toxic doses, whatever theculture conditions. Thus, depending on the culture conditions orC8 doses, C8 increases or decreases cell number and/or metabolicactivity. Moreover, C8 from 0.31 mM inhibited IL-1b-inducedsecretion of PGE2 by chondrocytes, corresponding to half of theIC50 on human GIIAPLA2 activity evaluated in vitro by enzymaticassay. Therefore, C8 could be a potent anti-inflammatory drug invivo. However, C8 did not inhibit IL-1b-induced NO secretion bychondrocytes cultured in DMEM alone or supplemented with0.1% BSA and slightly inhibited IL-1b-stimulated NO secretion inDMEM supplemented with 2% FCS. These data suggest that theanti-inflammatory property of C8 in chondrocytes mainly dependson its capacity to inhibit PLA2 activity.
COX-1 is involved in normal physiological functions, whereas COX-2 is involved in the inflammatory response. Anti-inflammatory drugs such as NSAIDs and selective COX-2 Figure 6. Effect of IL-1b and C8 on the COX-1, COX-2, and iNOS inhibitors, used to treat rheumatic disease, have severe side protein levels in articular chondrocytes. (A) Chondrocytes were effects owing to impairment in the balance between COX-1 and untreated or treated for 20 h with IL-1b alone or 1 h after the addition COX-2 [4,5,6]. Interestingly, the present work shows that the of C8 in DMEM. 20 mg aliquots of whole-cell protein extracts were potent PLA2 inhibitor C8 decreases PGE2 production without examined by western blot analysis with antibodies against COX-1, COX-2, and iNOS. a-tubulin immunodetection is shown as a control for impairing this balance. Consequently, C8 could be a useful protein loading and transfer. Results from one representative experi- candidate in developing new anti-inflammatory drugs lacking ment in five are shown. (B) Intensities of the COX-1 and COX-2 the side effects observed with NSAIDs and selective COX-2 immunoreactive bands evaluated by semi-quantitative scanning densitometry. Data represent the COX-1/COX-2 protein ratio and are In summary, we report on the design, synthesis and testing of 7 expressed as relative arbitrary units, where the IL-1b-treated group C1 analogs that differ from C1 by indole substitution or by indole represents 1. Values are means 6 SEM (n = 5 independent determina-tions). No significant differences were found between the groups.
replacement by other aromatic rings, the largest being phenan- threne. Compounds C2-C8 show both inhibitory activity onsecreted/synovial GIIAPLA2 and selectivity as compared withGIBPLA2, a pancreatic enzyme involved in the digestion of dietary which allows for only single-point computations of Poisson- phospholipids. The order of interaction energies predicted by Boltzmann solvation energies for the most stable minima of the molecular modeling of these compounds is associated with their molecular dynamics procedure. Our useful predictions made with experimental IC50 values with GIIAPLA2 used as a target. The the present simplified energy potential may be due to the very local most promising compound is C8 in terms of computed energy changes we made in the C1-C8 series. These bear on the series' balance for binding GIIAPLA2 and experimental potency towards sole terminal aromatic group and target a limited number of GIIAPLA2, namely one order of magnitude larger than that of amino acids, so that the accuracy of the energy potential may be C1. In addition, C8 is endowed with anti-inflammatory activity in sufficient. We plan to study such energy balances with the articular chondrocytes by inhibiting IL-1b-stimulated PGE2 polarizable molecular mechanics procedure SIBFA [30], which, secretion in these cells. Furthermore, it does not modify the ratio along with the Langlet-Claverie methodology for Continuum between the COX-1 and COX-2 isoenzymes. C8 is therefore an solvation [31], was recently used to investigate the binding of attractive candidate for anti-inflammatory therapy in joints.
inhibitors to metalloenzymes [32]. This study should also allow for Experiments in animal models of rheumatic diseases are in considering changes on other parts of the drugs as well.
progress in our laboratory.
One possible unfavorable feature of C8 is its enhanced lipophilicity as compared with the other compounds. Nevertheless, Materials and Methods this feature did not prevent the pharmacological efficiency of C8in chondrocytes. Reduction in Log P could be anticipated by Ethics Statements replacing phenantrene with heterocyclic analogs and/or substitu- Experimental protocols using rabbits complied with French tion with hydrophilic groups. Such reductions were seen on legislation on animal experimentation and were approved by PLoS ONE www.plosone.org June 2010 Volume 5 Issue 6 e10914 PLA2 Inhibitors in Chondrocyte INSERM (Intitut National de la Sante´ et de la Recherche Determination of PGE2 and nitrite concentrations in Me´dicale)'s Committee for Animal Studies.
20 h after the addition of IL-1b to the chondrocytes, culture Molecular modeling media were collected, and aliquots were stored at 280uC until Molecular modeling is described in Supporting Information S1.
PGE2 and nitrite quantification. PGE2 concentration in culturemedia was determined by use of an enzyme immunoassay (EIA) kit Synthesis of oxadiazolone derivatives (PGE2 EIA Kit-monoclonal; Cayman Chemical). Nitrite concen- Synthesis of compounds C1-C8 is described in Supporting tration was determined by a spectrophotometry method based on Information S1.
the Griess reaction [36]. Briefly, 200 mL of culture medium orsodium nitrite (NaNO2, Merck) standard dilutions were mixed In vitro PLA2 assay with 100 mL Griess reagent [0.5% (w/v) sulphanilic acid (Merck), Fatty-acid free BSA and pancreatic PLA 0.05% (w/v) N(1-naphtyl)ethylenediamine (Merck), 30% (v/v) 2 were from Sigma.
hGIIAPLA2 was prepared as previously described [33]. The acetic acid, 1.5 N HCl] and incubated for 10 min at 50uC. The fluorescent substrate for PLA2 assay, 1-hexadecanoyl-2-(10- absorbance was measured at 540 nm.
pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt(b-py-C10-PG) was from Molecular Probes (Eugene). PLA2 Preparation of whole-cell protein extracts, protein activity was evaluated as previously described [34] with b-py- quantification and western blot analysis C10-PG used as a substrate (2 mM final concentration). In a Proteins were extracted from the cultured cells by addition of total volume of 1 mL, the standard reaction medium contained lysis buffer [10 mM Tris (pH 7.4), 0.5% (v/v) NP40, 150 mM 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EGTA, NaCl, 1 mM PMSF, 0.1 mM Na3VO3, complete-EDTA-free 2 mM b-py-C10-PG, 0.1% fatty-acid free BSA and 6 ng/mL protease inhibitor cocktail (Roche)]. Cell lysates were centrifuged pancreatic PLA2 or 1 ng/mL hGIIAPLA2. The fluorescence for 15 min at 14000 rpm at 4uC and supernatants were collected.
(lex = 342 nm and lem = 398 nm) of the enzymatic reaction Protein concentrations were determined by the Bradford method medium was recorded for 3 min with use of a spectrofluorimeter [37] by use of the Protein Assay dye reagent (Bio-Rad). Protein LS 50 (Perkin-Elmer) equipped with a Xenon lamp. The extracts (20 mg) were size-separated by SDS-PAGE in a 10% (w/v) reaction was initiated by the addition of CaCl2 (10 mM, final polyacrylamide gel and electroblotted to a nitrocellulose mem- concentration). The increase in fluorescence was continuously brane. Equal protein loading and transfer was confirmed by recorded for 1 min, and PLA2 activity was calculated as staining the membrane with Ponceau Red [0.2% (w/v) in previously described [34]. When used, the inhibitor was added to the reaction medium after introduction of BSA. The activity 2O:acetic acid 99:1]. The membrane was sequentially incubated with antibodies against COX-1 (1:200, Santa Cruz Biotechnolo- is expressed in micromoles of fluorescent b-py-C10-PG hydro- gy), COX-2 (1:500, Santa Cruz Biotechnology), iNOS (1:400, BD lyzed per min. The standard error of the mean of three Biosciences) or a-tubulin (1:100, Santa Cruz Biotechnology) and independent experiments was less than 10%, which allows for then with peroxidase-conjugated donkey anti-goat IgG (1:20000) the determination of the IC50 values (concentration of inhibitors or donkey anti-rabbit IgG (1:200, both Santa Cruz Biotechnology).
producing 50% inhibition) of each compound.
Immunocomplexes were detected by an enhanced chemilumines-cence kit (Amersham Bioscience). The membrane was stripped by Isolation and culture of chondrocytes from rabbit incubation in 0.2 M NaOH between successive immunodetec- articular cartilage tions. Semi-quantitative scanning densitometry involved use of the Articular chondrocytes were isolated from 5-week-old Fauve de ImageJ program (NIH, USA).
Bourgogne female rabbits (CPA, Orleans, France) and cultured atthe first passage in conditions avoiding cell dedifferentiation as Statistical analysis previously described [35]. Cells were cultured at 37uC in 12-well Results are expressed as means 6 SEM for the number of plates in Ham's F-12 medium containing 10% FCS, 20 IU/mL experiments indicated. Statistical analysis involved use of the penicillin, and 20 mg/mL streptomycin (all from Invitrogen) until Kruskal-Wallis test, then the ANOVA Fisher's test. A P,0.05 was nearly confluent. Then medium was replaced with DMEM considered statistically significant.
(Invitrogen) containing 20 IU/mL penicillin, and 20 mg/mLstreptomycin and, if necessary, 0.1% fatty acid free BSA (Sigma)or 2% FCS. At this time the C8 compound dissolved in DMSO Supporting Information (Sigma) was added to the medium (the amount of DMSO was kept Supporting Information S1 Materials and Methods in chem- at 1% (v/v) in all the wells). 1 h after the addition of C8, IL-1b istry and molecular modeling.
(PeproTech) was added to the medium. Consequently, chondro- Found at: doi:10.1371/journal.pone.0010914.s001 (0.12 MB cytes were incubated for 20 h with IL-1b and for 21 h with C8.
Evaluation of cell viability At 18 h after the addition of IL-1b, 3-[4,5-dimethylthiazol-2- yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma) was added to We thank the computer center CINES-Montpellier for computer time and the cell culture medium at 0.5 mg/mL. Cells were incubated 2 technical support.
more hours at 37uC. The medium was then removed, and DMSOwas added to dissolve the formazan crystals. The absorbance of Author Contributions the resulting solution was spectrophotometrically measured at 570 Conceived and designed the experiments: JEO CZD FPR FH NG CC.
and 690 nm (background). The value corresponding to absorban- Performed the experiments: JEO NT SP LT FM AD EEH YS CC.
ce570nm - absorbance690nm was directly proportional to the number Analyzed the data: JEO NT CZD SP FPR FM NG CC. Wrote the paper: and activity of the viable cells.
JEO NT CZD FPR FM NG CC.
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