The impact of physicochemical and molecular properties in drug design: navigation in the "drug-like" chemical space.

Physicochemical and molecular properties influence both pharmacokinetic and pharmacodynamic process, as well as drug safety, often in a conflicting way. In this aspect the current trend in drug discovery is to consider ADME (T) properties in parallel with target affinity. The concept of "drug-likeness" defines acceptable boundaries of fundamental properties formulated as simple rules of thumb, in order to aid the medicinal chemist to prioritize drug candidates. Special attention is given to lipophilicity and molecular weight, since there is a tendency for those parameters to increase in regard to complex compounds generated by new technologies, with potential consequences in bioavailability, while high lipophilicity is also associated with undesired effects. Such rules have the advantage to be very simple and are easy to interpret; however their drawback is that they do not take into consideration uncertainties in measurements and calculations as well as the receptor requirements. The case of PPARs, a nuclear receptor family, is discussed in detail in regard to the chemical space covered by the ligands, focusing on the high demands of the ligand binding domain in both lipophilicity and molecular size. Such paradigms indicate that it would be more appropriate to adapt drug-like properties according to specific drug discovery projects.

Investigation of the retention behavior of structurally diverse drugs on alpha(1) acid glycoprotein column: insight on the molecular factors involved and correlation with protein binding data.

Retention of 49 structurally diverse drugs on alpha1 acid glycoprotein column was investigated under different chromatographic conditions. Acetonitrile and 2-propanol were used as organic modifiers at different percentages and the pH was adjusted at 7.0 using PBS. Analysis of extrapolated and isocratic retention in terms of lipophilicity and electrostatic interactions revealed significant effect of the nature and percentage of organic modifier, which was attributed to the different shielding degree of the charged sites on the stationary phase by the buffer constituents. AGP retention factors were compared to HSA retention factors analyzed previously. Application of LSER analysis, extended to incorporate fractions ionized, demonstrated hydrogen bond acidity, dipolarity/polarizability and excess molar refraction as the most significant parameters for all AGP chromatographic indices, elucidating the differentiation of AGP retention from octanol-water partitioning and HSA retention. An attempt to correlate AGP chromatographic indices to AGP association constants, available in literature, supported the importance of stationary shielding in retention mechanism. Thus, isocratic retention factors logk10(ACN)(AGP) show a moderate but still better performance than lipophilicity in the case of A variant and may be a useful tool for the estimation of relevant association constants. For F1/S binding simulation lower stationary phase shielding is needed to obtain a significant two term regression equation, where logk20(ACN)(AGP) exerts a secondary contribution next to the most important bulk effect expressed by molecular weight.

Octanol/water partitioning simulation by RP-HPLC for structurally diverse acidic drugs: comparison of three columns in the presence and absence of n-octanol as the mobile phase additive.

The advantageous effect of n-octanol as a mobile phase additive for lipophilicity assessment of structurally diverse acidic drugs both in the neutral and ionized form was explored. Two RP C18 columns, ABZ+ and Aquasil, were used for the determination of logkw indices, and the results were compared with those previously reported on a base-deactivated silica column. At pH 2.5, the use of n-octanol-saturated buffer as the mobile phase aqueous component led to high-quality 1:1 correlation between logkw and logP for the ABZ+ column, while inferior statistics were obtained for Aquasil. At physiological pH, the correlations were significantly improved if strongly ionized acidic drugs were treated separately from weakly ionized ones. In the latter case, 1:1 correlations between logD7.4 and logkw(oct) indices were obtained in the presence of 0.25% n-octanol. Concerning strongly ionized compounds, adequate correlations were established under the same conditions; however, slopes were significantly lower than unity, while large negative intercepts were obtained. According to the absolute difference (diff = logD7.4 ­ logkw) pattern, base-deactivated silica showed a better performance than ABZ+, however, the latter seems more efficient for the lipophilicity assessment of highly lipophilic acidic compounds. Aquasil may be the column of choice if logD7.4<3 with the limitation, however, that very hydrophilic compounds cannot be measured.

Analysis of PPAR-α/γ Activity by Combining 2-D QSAR and Molecular Simulation.

In the present study 2D-QSAR analysis was combined with information on crystallographic data and molecular modeling, in order to investigate dual PPAR-α/γ activity for a data set of 71 compounds, compiled from literature. Using Multivariate Data Analysis, satisfactory PLS models were generated for each receptor subtype separately. The models were based on simple and easily interpretable drug-like and constitutional descriptors, while the inclusion of MOLCONN-Z descriptors in the initial pool of variables had no considerable impact in model predictivity. By simultaneous analysis of both types of activity, a consensus PLS model for dual PPAR-α/γ activity could be derived, displaying the molecular features, which may lead to a balanced activity. All models were validated by permutation tests, by dividing the data set into training and test sets, as well as by external validation using a blind test set. Detailed inspection of PPAR-α and PPAR-γ crystal structures and molecular simulation supported the differentiation of most important descriptors in the separate PLS models, e.g. the higher impact of lipophilicity and bulk descriptors in PPAR-α and PPAR-γ activity respectively, as well as the effect of specific structural descriptors. Molecular simulation provided also explanation for the behavior of certain outliers in the PLS models.

The performance of 1-ethyl-3-methylimidazolium tetrafluoroborate ionic liquid as mobile phase additive in HPLC-based lipophilicity assessment.

Ionic liquids have been widely used as green alternative mobile phase additives to shield the residuals silanols groups and modify the stationary/mobile phase HPLC systems. The present study aimed to evaluate the performance of the ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4]) in producing extrapolated logk(w) indices suitable to substitute for octanol-water logP or logD values. The effect of [EMIM][BF4] was investigated for a set of basic and neutral drugs using two different columns, BDS and ABZ(+) . [EMIM][BF4] was added simply alone or in combination with n-octanol and was compared with the conventional masking agent n-decylamine. [EMIM][BF4] reduced the retention by suppressing silanophilic interactions, althoug to a lower extent than n-decylamine. Addition of n-octanol further decreased the retention by shielding silanol sites on BDS and/or interacting with polar groups through hydrogen bonding on ABZ(+) . Logk(w) /logD(7.4) relationships proved moderate compared with those derived upon addition of n-decylamine. They were considerably improved upon the introduction of protonated fraction F(+) in the correlation, reflecting ion pair formation between the chaotropic anion [BF4](-) and the protonated basic compounds. In this aspect, the ionic liquid [EMIM][BF4], although efficient as a masking agent, cannot be recommended as mobile phase additive to reproduce octanol-water partitioning.

Retention of structurally diverse drugs in human serum albumin chromatography and its potential to simulate plasma protein binding.

The retention behavior of 39 structurally diverse neutral, basic and acidic drugs was investigated on an HSA stationary phase using PBS buffer (pH 7.0) and acetonitrile or 2-propanol as organic modifiers. Extrapolated or directly measured logk(w) values as well as isocratic retention factors were correlated with plasma protein binding data taken from the literature. Retention factors determined in the presence of 10% acetonitrile led to high quality 1:1 correlation with apparent logK(HSA) values. The derived reference equation was successfully validated using a secondary set of 24 drugs. Further analysis of HSA retention into more fundamental properties revealed the involvement of anionic species in solute-stationary phase interactions, expressed by the negatively charged fraction, besides the partitioning mechanism which was reflected by lipophilicity. Protonation of basic drugs, although less important, may also influence retention, leading to reduced partitioning into the HSA surface as a net effect, while it seems to have no effect on HSA binding. The above results were further confirmed by linear solvation energy relationships (LSER).

Evaluation of FAK and Src expression in human benign and malignant thyroid lesions.

Focal Adhesion Kinase (FAK) and Src have been reported to regulate tumor growth, invasion, metastasis and angiogenesis. The present study aimed to evaluate by immunohistochemistry the clinical significance of FAK and Src expression in 108 patients with benign and malignant thyroid lesions. Total FAK expression provided a distinct discrimination between malignant and benign (p = 0.00001), as well as between papillary carcinoma and hyperplastic nodules thyroid lesions (p = 0.00005), being also associated with follicular cells' proliferative capacity (p = 0.0003). In malignant thyroid lesions, total FAK expression was associated with tumor size (p = 0.0455), and presence of capsular (p = 0.0102) and lymphatic (p = 0.0173) invasion. Total Src expression was borderline increased in cases of papillary carcinoma compared to hyperplastic nodules (p = 0.0993), being also correlated with tumor size (p = 0.0169). FAK and Src expression was ascribed to a significant extent to the phosphorylated forms of the enzymes, which provided a better discrimination between malignant and benign thyroid lesions. The current data revealed that FAK and to a lesser extent Src expression could be considered of clinical utility in thyroid neoplasia with potential use as therapeutic targets.

Direct injection liquid chromatography/positive ion electrospray ionization mass spectrometric quantification of methotrexate, folinic acid, folic acid and ondansetron in human serum.

A rapid liquid chromatography/positive ion electrospray mass spectrometric assay (LC/ESI-MS) was developed for the quantitation of methotrexate, folinic acid, folic acid and ondansetron in human serum. The assay was based on 100microL serum samples, following acetonitrile precipitation of proteins and filtration that enabled direct injection into the LC/MS system. All analytes and the internal standard, alfuzosin, were separated by using a Zorbax Eclipse XDB-C(8) analytical column (2.1mmx150.0mm i.d., particle size 3.5microm) with isocratic elution. The mobile phase was composed of a mixture of water/acetonitrile containing 0.1%, v/v formic acid (75:25, v/v), pumped at a flow rate of 0.15mLmin(-1). Quantitation of the analytes was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 0.01-25.00microgmL(-1) for methotrexate and 0.01-5.00microgmL(-1) for folic acid, folinic acid and ondansetron. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 7.0min for each sample made it possible to analyze a large number of human serum samples per day. The method can be used to quantify methotrexate, folinic acid, folic acid and ondansetron in human serum covering a variety of clinical studies and it was applied to the analysis of human serum samples obtained from children with acute lymphoblastic leukemia.

Application of quantitative structure-activity relationships for modeling drug and chemical transport across the human placenta barrier: a multivariate data analysis approach.

Pharmacological agents and environmental pollutants can transfer from mother to fetus across the placental barrier, leading to reproductive toxic effects. Ex vivo human placental perfusion constitutes the most widely used method to study placental transfer and metabolism of drugs and chemicals. The aim of the present study was to evaluate whether quantitative structure-activity relationship (QSAR) methodology could serve as an effective alternative tool to estimate drugs and chemicals transport across the human placental barrier on the basis of easily interpretable molecular, physicochemical and structural properties. Multivariate data analysis (MVDA) was applied to a set of 88 structurally diverse drugs and chemicals to model placental transfer expressed by clearance index values compiled from literature sources. An adequate and robust QSAR model (r(2) = 0.73, Q(2) = 0.71, RMSEE = 0.15) was established, providing an informative illustration of the contributing physicochemical, molecular and structural properties of the compounds in placental transfer process. Descriptors reflecting the polarity of compounds proved to be the most important with a negative sign. Lipophilicity and, at a lower extent, molecular size parameters exerted positive contribution in the model. Thus, QSAR analysis may be considered as a promising alternative tool to support high-throughput screening of drugs and chemicals in respect to their transport across placental barrier.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands as potential therapeutic agents to treat arthritis.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has already been considered as an attractive molecular target for the treatment of human metabolic disorders. Pleiotropic functions beyond this limit, such as anti-inflammatory and anti-proliferative effects against several pathological states, including atherosclerosis, osteoporosis and cancer, are currently being explored. Several natural and synthetic PPAR-gamma ligands have been the focus of extensive research effort as potent anti-inflammatory agents in diverse disease states. In the last decade, accumulative experimental evidence has further suggested that PPAR-gamma is involved in several inflammatory signaling pathways associated with arthritis. PPAR-gamma appears to be expressed by major cell populations in joints, such as chondrocytes, synoviocytes, fibroblasts and endothelial cells. PPAR-gamma ligands have also been shown to inhibit major inflammatory signaling pathways, reducing the synthesis of cartilage catabolic factors responsible for articular cartilage degradation in arthritis. In the present review the crucial role of PPAR-gamma ligands in arthritis and the underlying mechanisms participating in essential inflammatory signaling pathways are summarized. Taking into consideration the data so far, PPAR-gamma ligands seem to represent potential therapeutic agents in the aim to reduce mainly the inflammation implicated in arthritis. However, the precise molecular mechanisms through which PPAR-gamma ligands exert their actions are strongly recommended to be clarified, as both receptor-dependent and -independent actions were shown to be elicited.

Structural basis for the design of PPAR-gamma ligands: a survey on quantitative structure- activity relationships.

The present review after providing a short overview on PPARs and their pleiotropic action focuses on the QSAR studies reported mainly for PPAR-gamma agonists. The different 3D and 2D QSAR models are discussed, their impact in better understanding of the mechanism of action is analyzed and their contribution in the design of new molecules is outlined.

Diagnostic and prognostic utility of serum receptor-binding cancer antigen expressed on SiSo cells (RCAS1) levels in colon cancer patients.

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a human tumor-associated antigen that induces cell-cycle arrest and/or apoptosis in cells bearing the RCAS1 receptor. The aim of the present study was to elucidate the diagnostic and prognostic utility of RCAS1 levels in colon cancer patients. Serum RCAS1 levels were determined using a sandwich enzyme-linked immunosorbent assay in 97 colon cancer patients and 20 healthy individuals. The levels were significantly increased in colon cancer patients compared to healthy individuals (p<0.0001). Increased RCAS1 levels were significantly associated with advanced Dukes' stage (p=0.0079) and high histopathological tumor grade (p=0.0028). Univariate analysis revealed that colon cancer patients with elevated RCAS1 levels had significantly shorter overall survival times (log-rank test, p=0.027). By multivariate analysis, serum RCAS1 was identified as an independent prognostic factor (Cox regression analysis, p=0.033). In conclusion, colon cancer patients with advanced disease stage and grade and poor prognosis showed elevated serum RCAS1 levels. Assessment of serum RCAS1 levels could therefore be considered as a diagnostic and prognostic marker in colon neoplasia.

Quantitative structure-activity relationship (QSAR) methodology in forensic toxicology: modeling postmortem redistribution of structurally diverse drugs using multivariate statistics.

Postmortem redistribution (PMR) constitutes a multifaceted process, which renders the analytical results of drug concentrations inaccurate to be interpreted by forensic toxicologists. The aim of the present study was to evaluate whether quantitative structure-activity relationship (QSAR) methodology could serve as an effective tool to estimate the ability of drugs to redistribute across tissue barriers during postmortem period on the basis of their molecular, physicochemical and structural properties. In this aspect, multivariate data analysis (MVDA) was applied to a set of 77 structurally diverse drugs. PMR data expressed by the central:peripheral concentration ratio (C:P ratio) was taken from the literature. An adequate and robust QSAR model (R(2)=0.65, Q(2)=0.56, RMSEE=0.34) was established for 59 (77%) out of 77 drugs. Although the derived QSAR model presented limited applicability, it provided an informative illustration of the contributing molecular, physicochemical and structural properties in PMR process. Drugs with strong basic properties and enhanced molecular size, flexibility, lipophilicity and number of halogens were found to be susceptible to increased PMR. Due to the high complexity of PMR process, further QSAR studies need to focus on structurally related drugs to develop more specific models, which could serve as alternative tools to evaluate PMR for different chemical classes.

Involvement of hepatic stimulator substance in experimentally induced fibrosis and cirrhosis in the rat.

Liver fibrosis results from sustained wound healing response to chronic liver injury. Liver cirrhosis, the end stage of the fibrotic process, is characterized by disruption of the entire liver architecture and reduced hepatocyte regenerative ability. Hepatic stimulator substance (HSS) is a liver-specific growth factor triggering hepatocyte proliferation in vitro and in vivo. Previous studies have indicated the involvement of HSS in animal models of acute liver injury. The aim of the present study was to investigate the involvement of HSS in the process of fibrosis and cirrhosis induction. Liver fibrosis and cirrhosis were induced in rats by thioacetamide (TAA) administration (300 mg/l) in the drinking water for 3 months, and animals were killed at 0, 1, 2, and 3 months of treatment. TAA administration resulted in progressively increasing liver fibrosis, leading to the onset of cirrhosis at the end of the experimental time. HSS was continuously produced during the course of fibrosis and cirrhosis induction, peaking at the 2nd month of TAA treatment, coinciding with markers of hepatic proliferative capacity, as thymidine kinase activity and DNA biosynthesis. Significantly reduced HSS activity was noted in cirrhotic liver (3rd month). In this case, the exogenous HSS administration during the 3rd month of TAA treatment suppressed the onset of liver cirrhosis, stimulating the hepatic regenerative capacity. Our data indicate the active participation of HSS in the process of fibrosis and cirrhosis induction post-TAA treatment in rats, suggesting also the beneficial effect of HSS treatment against cirrhosis induction with future possible clinical implications.

Chromatographic behavior of zwitterionic enalapril-exploring the conditions for lipophilicity assessment.

The chromatographic behavior of enalapril was investigated under different stationary and mobile phase conditions in an effort to unravel interferences in the underlying retention mechanism, which would affect its relation to octanol-water partitioning. Extrapolated retention factors, logk(w), were used as relevant chromatographic indices. The retention/pH profile was established and the peak split phenomenon, associated with cis/trans interconversion, was also monitored as a function of pH. The pH at maximum retention and minimum peak split occurrence was chosen for further investigation, so that the presence of zwitterionic structure was guaranteed and any effect of cis/trans interconversion could be ignored. Retention of zwitterionic enalapril was found to be very sensitive to mobile phase conditions in regard to organic modifier as well to the aqueous component. The use of morpholine-propanesulfonic acid (MOPS) as buffer and the presence of n-octanol as mobile phase additive proved critical factors for maximum suppression of secondary interactions. Nevertheless, the corresponding extrapolated retention factor was considerably larger than octanol-water logD value at the isoelectric point. However, logk(w) could be successfully converted to logD by means of a calibration equation established for ionized acidic compounds.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands: novel pharmacological agents in the treatment of ischemia reperfusion injury.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands constitute important insulin sensitizers that have already been used for the treatment of human metabolic disorders, exerting also pleiotropic effects on inflammatory related diseases and cancer. Ischemia-reperfusion injury that is mainly associated with organ transplantation constitutes a serious complication with a great relevance in clinical practice. Accumulating experimental data have recently revealed that natural and synthetic PPAR-gamma ligands exert beneficial effects against ischemia-reperfusion injury. The present review summarizes the available information on the role of PPAR-gamma ligands in ischemia-reperfusion injury amongst the different organ systems. Taking into consideration the data so far, PPAR-gamma ligands seem to represent potential therapeutic agents in the aim to reduce or even prevent injury associated with ischemia-reperfusion.

Quantitative structure-activity relationships for PPAR-gamma binding and gene transactivation of tyrosine-based agonists using multivariate statistics.

Peroxisome proliferator-activated receptor-gamma offers a molecular target for drugs aimed to treat type II diabetes mellitus, while its therapeutic potency against cancer disease is currently being explored in preclinical studies. Tyrosine derivatives constitute a major class of peroxisome proliferator-activated receptor-gamma agonists attracting considerable research interest in drug discovery. Thus, the establishment of adequate QSAR models would serve as a guide for further molecular design. In the present study, multivariate data analysis was applied on a large set of tyrosine-based peroxisome proliferator-activated receptor-gamma agonists for modelling binding affinity, expressed as pKi and gene transactivation, expressed as pEC(50). A pool of descriptors based on physicochemical and molecular properties as well as on specific structural characteristics was used and two PLS models with satisfactory statistics were produced for binding data. According to them, molecular weight, rotatable bonds and lipophilicity were found to exert a considerable positive influence, while excess negative and positive charge created by additional acidic or basic groups in the molecules was unfavourable. With gene transactivation data, an adequate model was obtained only for the highly active compounds if considered separately. The higher complexity incorporated in gene transactivation data was further investigated by establishing a PLS model, which improved the inter-relationship between pEC(50) and pKi.

PPAR-gamma signaling pathway in placental development and function: a potential therapeutic target in the treatment of gestational diseases.

BACKGROUND: PPAR-gamma is a target for the treatment of metabolic disorders, as Pioglitazone and Rosiglitazone are already used against type 2 diabetes. Pleiotropic functions, such as antiproliferative and anti-inflammatory effects against several pathological states, including cardiovascular disease and cancer, are currently being explored in clinical studies. OBJECTIVE: Evidence indicates that PPAR-gamma is expressed in the placenta, playing a crucial role in placental development and function, while PPAR-gamma ligands appear to modulate fetal membrane signals. Thus, in the last few years, the pivotal role of PPAR-gamma in placental biology has been the focus of extensive research, as diabetes appears to be the most common metabolic dysfunction in pregnancy. METHODS: We aim to present data concerning the expression of PPAR-gamma in animal and human placenta, underlining its significance in normal placental development and several gestational diseases. The effects of PPAR-gamma ligands as modulators of placental biology in normal and certain pathological conditions are also discussed. RESULTS/CONCLUSION: Current research provides substantial evidence that PPAR-gamma plays a pivotal role in placental biology and may reveal new perspectives in the treatment of gestational diseases.

Coxsackievirus and adenovirus receptor expression in human endometrial adenocarcinoma: possible clinical implications.

The coxsackievirus and adenovirus receptor (CAR) is a crucial receptor for the entry of both coxsackie B viruses and adenoviruses into host cells. CAR expression on tumor cells was reported to be associated with their sensitivity to adenoviral infection, while it was considered as a surrogate marker for monitoring and/or predicting the outcome of adenovirus-mediated gene therapy. The aim of the present study was to evaluate the clinical significance of CAR expression in endometrial adenocarcinoma. CAR expression was assessed immunohistochemically in tumoral samples of 41 endometrial adenocarcinoma patients and was statistically analyzed in relation to various clinicopathological parameters, tumor proliferative capacity and patient survival. CAR positivity was noted in 23 out of 41 (56%) endometrial adenocarcinoma cases, while high CAR expression in 8 out of 23 (35%) positive ones. CAR intensity of immunostaining was classified as mild in 11 (48%), moderate in 10 (43%) and intense in 2 (9%) out of the 23 positive cases. CAR positivity was significantly associated with tumor histological grade (p = 0.036), as well differentiated tumors more frequently demonstrating no CAR expression. CAR staining intensity was significantly associated with tumor histological type (p = 0.016), as tumors possessing squamous elements presented more frequently intense CAR immunostaining. High CAR expression showed a trend to be correlated with increased tumor proliferative capacity (p = 0.057). Patients with tumors presenting moderate or intense CAR staining intensity were characterized by longer survival times than those with mild one; however, this difference did not reach statistical significance. These data reveal, for the first time, the expression of CAR in clinical material obtained from patients with endometrial adenocarcinoma in relation to important clinicopathological parameters for their management. As CAR appears to modulate the proliferation and characteristics of cancer cells, its expression could be considered of possible clinical importance for future (gene) therapy applications.

Alternative measures of lipophilicity: from octanol-water partitioning to IAM retention.

This review describes lipophilicity parameters currently used in drug design and QSAR studies. After a short historical overview, the complex nature of lipophilicity as the outcome of polar/nonpolar inter- and intramolecular interactions is analysed and considered as the background for the discussion of the different lipophilicity descriptors. The first part focuses on octanol-water partitioning of neutral and ionisable compounds, evaluates the efficiency of predictions and provides a short description of the experimental methods for the determination of distribution coefficients. A next part is dedicated to reversed-phase chromatographic techniques, HPLC and TLC in lipophilicity assessment. The two methods are evaluated for their efficiency to simulate octanol-water and the progress achieved in the refinement of suitable chromatographic conditions, in particular in the field of HPLC, is outlined. Liposomes as direct models of biological membranes are examined and phospolipophilicity is compared to the traditional lipophilicity concept. Difficulties associated with liposome-water partitioning are discussed. The last part focuses on Immobilised Artificial Membrane (IAM) chromatography as an alternative which combines membrane simulation with rapid measurements. IAM chromatographic retention is compared to octanol-water and liposome-water partitioning as well as to reversed-phase retention and its potential to predict biopartitioning and biological activities is discussed.