Sensitivity of Different In Vitro Performance Tests and Their In Vivo Relevance for Calcipotriol/Betamethasone Ointment.

PURPOSE: We compared results of in vitro performance testing with results of therapeutic equivalence study for calcipotriol/betamethasone ointment, to evaluate their sensitivity and in vivo relevance. METHODS: Different in vitro methods were used to evaluate drug release and permeation from the test and reference ointment. Moreover, 444 psoriasis patients were randomized in the therapeutic equivalence study and the parameters of efficacy and safety were compared with in vitro results. RESULTS: In vitro release and permeation rate of calcipotriol and betamethasone from the test formulation was higher than from the reference product for all methods used (p ≤ 0.05 for calcipotriol and p < 0.01 for betamethasone). Observed batch-to-batch variability of reference product confirmed high sensitivity and discriminatory power of in vitro methods. Higher release and permeation rate of calcipotriol and betamethasone from test product was reflected in the efficacy assessment (mean response difference 4.78 mPASI percentage points), but the observed difference was within the equivalence margins. Systemic exposure to calcipotriol and betamethasone was similar in both treatment groups. CONCLUSION: The results of in vitro experiments rank orderly correlated with the results of clinical study. In vitro methods are more sensitive and highly discriminatory when compared to in vivo performance.

Different Culture Conditions Affect Drug Transporter Gene Expression, Ultrastructure, and Permeability of Primary Human Nasal Epithelial Cells.

PURPOSE: This study aimed to characterize a commercially available primary human nasal epithelial cell culture and its gene expression of a wide range of drug transporters under different culture conditions. METHODS: Human nasal cells were cultured in three different types of culture media at the air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 wks. The effects of the different cell culture conditions were evaluated using light and electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran, and gene expression profiling of 84 drug transporters. RESULTS: The type of culture medium affected cell ultrastructure, TEER, and dextran permeation across epithelia. The expression of 20 drug transporter genes depended on the culture interface and/or time in culture; the A-L interface and longer time in culture favored higher expression levels of five ABC and seven SLC transporters. CONCLUSIONS: Culture conditions influence the morphology, barrier formation, permeation properties, and drug transporter expression of human nasal epithelial cells, and this must be taken into consideration during the establishment and validation of in vitro models. A thorough characterization of a nasal epithelial model and its permeability properties is necessary to obtain an appropriate standardized model for the design of aerosol therapeutics and drug transport studies.

High-throughput automated dissolution method applicable for a wide dose range of controlled release pellets.

The aim of the present study was to demonstrate the application of an automated high-throughput (HT) dissolution method as a useful screening tool for characterization of controlled release pellets in the formulation development phase. Five controlled release pellet formulations with drug substances exhibiting high or low solubility were chosen to investigate the correlation of the automated HT dissolution method with the conventional dissolution testing. Overall, excellent correlations (R(2 )>( )0.96) between the HT and the conventional dissolution method were obtained. In one case the initial unsatisfactory correlation (R(2 )=( )0.84) and poor method agreement (SD = 12.5) was improved by optimizing the HT dissolution method with design of experiment approach. Here in comparison to initial experimental HT dissolution settings, increased amount of pellets (25% of the capsule filling mass), lower temperature (22 °C) and no shaking resulted in significantly better correlation (R(2 )=( )0.97) and method agreement (SD = 5.3). These results show that such optimization is valuable for the development of HT dissolution methods. In conclusion, the high correlation of dissolution profiles obtained from the conventional and the automated HT dissolution method combined with low within-sample and measurement system variability, justifies the utilization of the automated HT dissolution method during development phase of controlled release pellets.

The characterization of the human nasal epithelial cell line RPMI 2650 under different culture conditions and their optimization for an appropriate in vitro nasal model.

PURPOSE: The further characterization of the cell line RPMI 2650 and the evaluation of different culture conditions for an in vitro model for nasal mucosa. METHODS: Cells were cultured in media MEM or A-MEM at air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 weeks. Different cryopreservation methods and cell culture techniques were evaluated with immunolabelling of junctional proteins, ultrastructural analysis using electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran and jacalin, and gene expression profiling of 84 drug transporters. RESULTS: Cell proliferation and differentiation depended on the used medium. The established epithelia expressed occludin, claudin-1, and E-cadherin under all conditions. Cells grown at the A-L interface formed more layers and exhibited a higher TEER and lower dextran and jacalin permeability than at the L-L interface, where cells morphologically exhibited a more differentiated phenotype. The expression of ABC and SLC transporters depended on culture duration and interface. CONCLUSIONS: The RPMI 2650 cells form a polarized epithelium resembling nasal mucosa. However, different culture conditions have a significant effect on cell ultrastructure, barrier integrity, and gene expression, and should be considered when using this cell line as an in vitro model for drug permeability studies and screening of nasal drug candidates.

Slightly viscous dispersions of mucoadhesive polymers as vehicles for nasal administration of dopamine and grape seed extract-loaded solid lipid nanoparticles.

With the aim to find an alternative vehicle to the most used thermosensitive hydrogels for efficient nanotechnology-based nose-to-brain delivery approach for Parkinson's disease (PD) treatment, in this work we evaluated the Dopamine (DA) and the antioxidant grape seed-derived pro-anthocyanidins (Grape Seed Extract, GSE) co-loaded solid lipid nanoparticles (SLNs) put in slight viscous dispersions (SVDs). These SVDs were prepared by dispersion in water at low concentrations of mucoadhesive polymers to which SLN pellets were added. For the purpose, we investigated two polymeric blends, namely Poloxamer/Carbopol (PF-127/Carb) and oxidized alginate/Hydroxypropylmethyl cellulose (AlgOX/HPMC). Rheological studies showed that the two fluids possess Newtonian behaviour with a viscosity slightly higher that water. The pH values of the SVDs were mainly within the normal range of nasal fluid as well as almost no osmotic effect was associated to both SVDs. All the SVDs were capable to provide DA permeation through nasal porcine mucosa. Moreover, it was found that PF-127/Carb blend possesses penetration enhancer capability better than the Alg OX/HPMC combination. Flow cytometry studies demonstrated the uptake of viscous liquids incorporating fluorescent SLNs by human nasal RPMI 2650 cell in time-dependent manner. In conclusion, the SVD formulations may be considered promising alternatives to thermosensitive hydrogels strategy. Moreover, in a broader perspective, such SVD formulations may be also hopeful for treating various neurological diseases beyond PD treatment.

Insights into subtle conformational differences in the substrate-binding loop of fungal 17ß-hydroxysteroid dehydrogenase: a combined structural and kinetic approach.

The 17ß-HSD (17ß-hydroxysteroid dehydrogenase) from the filamentous fungus Cochliobolus lunatus (17ß-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyses the interconversion of inactive 17-oxo-steroids and their active 17ß-hydroxy counterparts. 17ß-HSDcl belongs to the SDR (short-chain dehydrogenase/reductase) superfamily. It is currently the only fungal 17ß-HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH-dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provide insights into the evolution of the HSD enzymes. The crystal structures of the 17ß-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active-site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that probably modulate the catalytic activity of 17ß-HSDcl. Coumestrol, a plant-derived non-steroidal compound with oestrogenic activity, inhibits 17ß-HSDcl [IC50 2.8 µM; at 100 µM substrate (4-oestrene-3,17-dione)] by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is stabilized further by π-π stacking interactions with Tyr212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and, in addition to the low steroid solubility, it prevents the accumulation of enzyme-coenzyme-steroid ternary complexes.

Applicability of RPMI 2650 and Calu-3 Cell Models for Evaluation of Nasal Formulations.

The RPMI 2650 and Calu-3 cell lines have been previously evaluated as models of the nasal and airway epithelial barrier, and they have demonstrated the potential to be used in drug permeation studies. However, limited data exist on the utilization of these two cell models for the assessment of nasal formulations. In our study, we tested these cell lines for the evaluation of in vitro permeation of intranasally administered drugs having a local and systemic effect from different solution- and suspension-based formulations to observe how the effects of formulations reflect on the measured in vitro drug permeability. Both models were shown to be sufficiently discriminative and able to reveal the effect of formulation compositions on drug permeability, as they demonstrated differences in the in vitro drug permeation comparable to the in vivo bioavailability. Good correlation with the available bioavailability data was also established for a limited number of drugs formulated as intranasal solutions. The investigated cell lines can be applied to the evaluation of in vitro permeation of intranasally administered drugs with a local and systemic effect from solution- and suspension-based formulations.

In Vitro Ciliotoxicity and Cytotoxicity Testing of Repeated Chronic Exposure to Topical Nasal Formulations for Safety Studies.

Certain active drugs and excipients of nasal formulations may impair ciliary function and mucociliary clearance. The ciliary beat frequency (CBF) is a key parameter for determining mucociliary clearance rate, and in vitro assessments of CBF have proven to be accurate and reproducible. Since topical nasal formulations are applied with repeated doses, it is essential to elucidate their chronic, as opposed to acute, effect on mucociliary clearance and nasal mucosa. The aim of this study was to assess for the first time the ciliotoxicity and cytotoxicity of nasal sprays intended for chronic treatment (with repeated doses) using a previously designed set-up for CBF measurements. For 2 weeks, the 3D nasal MucilAir™ in vitro models were treated daily with undiluted or clinically relevant doses of mometasone nasal spray, placebo nasal spray, culture medium, or they were untreated. We demonstrated a dose-dependent and time-dependent (cumulative) effect of the nasal sprays on ciliary activity and cytotoxicity using CBF measurements and ultrastructural analysis, respectively. Our results indicate that repeated administration of clinically relevant doses of mometasone nasal spray is safe for in vivo use, which is in good agreement with a previous clinical study. Overall, our study suggests that such in vitro assays have great potential for topical nasal drug screening.

Prediction of pharmacokinetic studies outcome for locally acting nasal sprays by using different in vitro methods.

Demonstration of bioequivalence of locally acting nasal spray formulations is a challenging task and the regulatory agencies have different approach towards this goal. The pharmacokinetic bioequivalence studies are recognized as necessary for assessment of equivalent systemic exposure. We utilized three different in vitro methods for nasal spray evaluation and compared those results with the results of pharmacokinetic studies of different formulations of four intranasal corticosteroids, in order to evaluate their in vivo relevance. Two cell lines, RPMI 2650 and Calu-3, Transwell® polycarbonate membranes with different pore size and lipid-oil-lipid tri-layer membrane in the parallel artificial membrane permeability assay (PAMPA) system were used for this purpose. The in vitro results correlated with the results of pharmacokinetic studies and correctly predicted (non)equivalence of the nasal sprays, showing that in vitro methods are good indicator of the in vivo outcome. The Transwell® and PAMPA in vitro methods were additionally implemented for testing batch-to-batch variability of reference nasal spray formulations. The results from the Transwell® assay for the two poorly soluble corticosteroids are possibly over-discriminatory in showing differences between batches of reference nasal sprays. Overall, the three in vitro methods have potential to predict the results of bioequivalence testing of nasal spray products.

Standardization of esophageal adenocarcinoma in vitro model and its applicability for model drug testing.

FLO-1 cell line represents an important tool in esophageal adenocarcinoma (EAC) research as a verified and authentic cell line to study the disease pathophysiology and antitumor drug screenings. Since in vitro characteristics of cells depend on the microenvironment and culturing conditions, we performed a thorough characterization of the FLO-1 cell line under different culturing conditions with the aim of (1) examining the effect of serum-free growth medium and air-liquid interface (A-L) culturing, which better reflect physiological conditions in vivo and (2) investigating the differentiation potential of FLO-1 cells to mimic the properties of the in vivo esophageal epithelium. Our study shows that the composition of the media influenced the morphological, ultrastructural and molecular characteristics of FLO-1 cells, such as the expression of junctional proteins. Importantly, FLO-1 cells formed spheres at the A-L interface, recapitulating key elements of tumors in the esophageal tube, i.e., direct contact with the gas phase and three-dimensional architecture. On the other hand, FLO-1 models exhibited high permeability to model drugs and zero permeability markers, and low transepithelial resistance, and therefore poorly mimicked normal esophageal epithelium. In conclusion, the identified effect of culture conditions on the characteristics of FLO-1 cells should be considered for standardization, data reproducibility and validity of the in vitro EAC model. Moreover, the sphere-forming ability of FLO-1 cells at the A-L interface should be considered in EAC tumor biology and anticancer drug studies as a reliable and straightforward model with the potential to increase the predictive efficiency of the current in vitro approaches.

Suitability and functional characterization of two Calu-3 cell models for prediction of drug permeability across the airway epithelial barrier.

The Calu-3 cell line has been largely investigated as a physiological and pharmacological model of the airway epithelial barrier. Its suitability for prediction of drug permeability across the airway epithelia, however, has not been yet evaluated by using large enough set of model drugs. We evaluated two Calu-3 cell models (air-liquid and liquid-liquid) for drug permeability prediction based on the recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. Bidirectional permeability assays using 22 model drugs and several zero permeability markers, as well as using ABC transporter substrates were conducted. Functional activity of P-gp, but not of BCRP was revealed. The potential of the Calu-3 cells to be used as a model of the nasal epithelial barrier, despite their different anatomical origin, has been demonstrated by the obtained excellent correlation with the fully differentiated 3D human nasal epithelial model (MucilAir™) for 11 model drugs, as well as by the good correlation obtained with the human nasal epithelial cell line RPMI 2650. In addition, the permeability values determined in the two Calu-3 models correlated well with the intestinal permeability model Caco-2.

Ciliary beat frequency of in vitro human nasal epithelium measured with the simple high-speed microscopy is applicable for safety studies of nasal drug formulations.

Nasal drug formulations can be effective for local delivery of therapeutic drugs to the sinonasal mucosa or for systemic drug delivery by absorption directly into the bloodstream. The growing field of potential nasal therapies includes nasal vaccination and even treatment of neurodegenerative diseases. However, it is important that nasal drug formulations don't have a disruptive effect on the cilia and mucosa of nasal epithelium. Mucociliary clearance represents the first host defence of the respiratory tract that requires the coordinated beating of cilia. A key parameter to determine mucociliary clearance is ciliary beat frequency (CBF). The objective of this study was to validate the high-speed digital imaging for CBF measurements in nasal MucilAir™ in vitro model and to test its potential for ciliotoxicity studies to evaluate the safety of investigational nasal drug formulations. Our CBF measuring setup was first validated by benzalkonium chloride, a common-practice preservative with cilio-inhibiting effect. Next, MucilAir™ model was treated with mometasone nasal spray (Mommox®/Mometasone Sandoz®). Short term cilio-stimulatory effect and dose dependent effect of mometasone nasal spray were demonstrated. Post-treatment analysis showed un-altered ultrastructure of MucilAir™ model. In conclusion, characterization of the ciliary activity of nasal MucilAir™ in vitro model and its response to relevant agents with herein developed efficient and reproducible set up for CBF analysis show great potential of this model for airway ciliotoxicity studies.

New high-throughput fluorimetric assay for discovering inhibitors of UDP-N-acetylmuramyl-L-alanine: D-glutamate (MurD) ligase.

A novel assay for monitoring the activity of the bacterial enzyme UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD ligase) is presented. MurD, which belongs to an enzyme family of Mur ligases, is essential for the synthesis of bacterial peptidoglycan and therefore represents an attractive target for the discovery of novel antibacterial agents. The inhibition assay described in this article is amenable to high-throughput screening. It is based on the detection of the accumulation of adenosine 5'-diphosphate (ADP), a product of the reaction catalyzed by MurD ligase, by conversion to a fluorescent signal via a coupled enzyme system, using the ADP Quest assay kit from DiscoveRx. The novel assay has been validated by obtaining KM,app values for substrates D-Glu, UDP- N-acetylmuramyl-L-alanine (UMA) and ATP that are in agreement with the data reported in the literature. A counterscreen assay was introduced to eliminate false positives, and some of the known MurD inhibitors have been retested to compare the data measured with different methods. Moreover, a focused library of around 1000 compounds was screened for the inhibition of MurD to assess the performance and robustness of the assay. Finally, a novel MurD inhibitor belonging to a new structural class, with an IC50 value of 105 microM, was discovered.

Mutations that affect coenzyme binding and dimer formation of fungal 17beta-hydroxysteroid dehydrogenase.

The 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/reductase superfamily, and it functions as a dimer that is composed of two identical subunits. By constructing the appropriate mutants, we have examined the M204 residue that is situated in the coenzyme binding pocket, for its role in the binding of the coenzyme NADP(H). We have also studied the importance of hydrophobic interactions through F124, F132, F133 and F177 for 17beta-HSDcl dimer formation. The M204G substitution decreased the catalytic efficiency of 17beta-HSDcl, suggesting that M204 sterically coerces the nicotinamide moiety of the coenzyme into the appropriate position for further hydride transfer. Phenylalanine substitutions introduced at the dimer interface produced inactive aggregates and oligomers with high molecular masses, suggesting that these hydrophobic interactions have important roles in the formation of the active dimer.

Suitability of RPMI 2650 cell models for nasal drug permeability prediction.

The RPMI 2650 cell line has been a subject of evaluation as a physiological and pharmacological model of the nasal epithelial barrier. However, its suitability for drug permeability assays has not yet been established on a sufficiently large set of model drugs. We investigated two RPMI 2650 cell models (air-liquid and liquid-liquid) for nasal drug permeability determination by adopting the most recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. The permeability of 23 model drugs and several zero permeability markers across the cell models was assessed. The functional expression of two efflux transporters P-glycoprotein (P-gp) and Breast Cancer Resistant Protein (BCRP) was shown to be negligible by bidirectional transport studies using appropriate transporter substrates and inhibitors. The model drug permeability determined in the two RPMI 2650 cell models was correlated with the fully differentiated nasal epithelial model (MucilAir™). Additionally, correlations between the drug permeability in the investigated cell models and the ones determined in the Caco-2 cells and isolated rat jejunum were established. In conclusion, the air-liquid RPMI 2650 cell model is a promising pharmacological model of the nasal epithelial barrier and is much more suitable than the liquid-liquid model for nasal drug permeability prediction.

Demonstrating suitability of the Caco-2 cell model for BCS-based biowaiver according to the recent FDA and ICH harmonised guidelines.

OBJECTIVE: According to the regulatory guidelines, one of the critical steps in using in-vitro permeability methods for permeability classification is to demonstrate the suitability of the method. Here, suitability of the permeability method by using a monolayer of cultured epithelial cells was verified with different criteria. METHODS: Imaging with a transmission electron microscope was used for characterisation of the cells. Monolayer integrity was confirmed by transepithelial electrical resistance measurements and permeability of zero permeability marker compounds. Real-time polymerase chain reaction was employed to evaluate expression levels of 84 known transporters. Samples for bidirectional permeability determination were quantified by ultra-performance liquid chromatography. KEY FINDINGS: The Caco-2 cells grow in an intact monolayer and morphologically resemble enterocytes. Genes of 84 known transporters were expressed at different levels; furthermore, expression was time depended. Functional expression of efflux transporter P-glycoprotein was confirmed. We established a correlation between permeability coefficients of 21 tested drug substances ranging from low, moderate and high absorption with human fraction absorbed literature data (R2  = 0.84). CONCLUSIONS: Assay standardisation assures the consistency of experimental data. Only such fully characterised model has the ability to accurately predict drug's intestinal permeability at the early stage of research or for the BCS-based biowaiver application.

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase.

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.

His164 regulates accessibility to the active site in fungal 17beta-hydroxysteroid dehydrogenase.

17beta-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/ reductase superfamily. To study the catalytic properties of this enzyme, we prepared several specific mutations of 17beta-HSDcl (Tyr167Phe, His164Trp/Gly, Tyr212Ala). Wild-type 17beta-HSDcl and the 17beta-HSDcl mutants were evaluated by chromatographic, kinetic and thermodynamic means. The Tyr167Phe mutation resulted in a complete loss of enzyme activity, while substitution of His164 with Trp and Gly both resulted in higher specificity number (V/K) for the steroid substrates, which are mainly a consequence of easier accessibility of steroid substrates to the active-site hollow under optimized conditions. The Tyr212Ala mutant showed increased activity in the oxidative direction, which appears to be a consequence of increased NADPH dissociation. The kinetic characterizations and thermodynamic analyses also suggest that His164 and Tyr212 in 17beta-HSDcl have a role in the opening and closing of the active site of this enzyme and in the discrimination between oxidized and reduced coenzyme.

Rational design of novel mutants of fungal 17beta-hydroxysteroid dehydrogenase.

Reduction of 17-ketosteroids is a biocatalytic process of economic significance for the production of steroid drugs. This reaction can be catalyzed by different microbial 17beta-hydroxysteroid dehydrogenases (17beta-HSD), like the 17beta-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3beta,17beta-HSD from Pseudomonas testosteroni. In addition to the bacterial 3beta,17beta-HSD the 17beta-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17beta-HSD that has been expressed as a recombinant protein and fully characterized. On the basis of its modeled 3D structure, we selected several positions for the replacement of amino acids by site-directed mutagenesis to change substrate specificity, alter coenzyme requirements, and improve overall catalytic activity. Replacement of Val161 and Tyr212 in the substrate-binding region by Gly and Ala, respectively, increased the initial rates for the conversion of androstenedione to testosterone. Replacement of Tyr49 within the coenzyme binding site by Asp changed the coenzyme specificity of the enzyme. This latter mutant can convert the steroids not only in the presence of NADP(+) and NADPH, but also in the presence of NADH and NAD(+). The replacement of His164, located in the non-flexible part of the 'lid' covering the active center resulted in a conformation of the enzyme that possessed a higher catalytic activity.

Structural basis for inhibition of 17ß-hydroxysteroid dehydrogenases by phytoestrogens: The case of fungal 17ß-HSDcl.

Phytoestrogens are plant-derived compounds that functionally and structurally mimic mammalian estrogens. Phytoestrogens have broad inhibitory activities toward several steroidogenic enzymes, such as the 17ß-hydroxysteroid dehydrogenases (17ß-HSDs), which modulate the biological potency of androgens and estrogens in mammals. However, to date, no crystallographic data are available to explain phytoestrogens binding to mammalian 17ß-HSDs. NADP(H)-dependent 17ß-HSD from the filamentous fungus Cochliobolus lunatus (17ß-HSDcl) has been the subject of extensive biochemical, kinetic and quantitative structure-activity relationship studies that have shown that the flavonols are the most potent inhibitors. In the present study, we investigated the structure-activity relationships of the ternary complexes between the holo form of 17ß-HSDcl and the flavonols kaempferol and 3,7-dihydroxyflavone, in comparison with the isoflavones genistein and biochanin A. Crystallographic data are accompanied by kinetic analysis of the inhibition mechanisms for six flavonols (3-hydroxyflavone, 3,7-dihydroxyflavone, kaempferol, quercetin, fisetin, myricetin), one flavanone (naringenin), one flavone (luteolin), and two isoflavones (genistein, biochanin A). The kinetics analysis shows that the degree of hydroxylation of ring B significantly influences the overall inhibitory efficacy of the flavonols. A distinct binding mode defines the interactions between 17ß-HSDcl and the flavones and isoflavones. Moreover, the complex with biochanin A reveals an unusual binding mode that appears to account for its greater inhibition of 17ß-HSDcl with respect to genistein. Overall, these data provide a blueprint for identification of the distinct molecular determinants that underpin 17ß-HSD inhibition by phytoestrogens.