QUANTITATIVE STRUCTURE–PHARMACOKINETICS RELATIONSHIP FOR PLASMA PROTEIN BINDING OF NEUTRAL DRUGS

Objective: Plasma protein binding (PPB) of drugs is important pharmacokinetic (PK) phenomena controlling the free drug concentration in plasma and the overall PK and pharmacodynamic profile. Prediction of PPB at the very early stages of drug development process is of paramount importance for the success of new drug candidates. The study presents a quantitative structure–pharmacokinetics relationship (QSPkR) modelling of PPB for neutral drugs.Methods: The dataset consists of 117 compounds, described by 138 molecular descriptors. Genetic algorithm and stepwise multiple linear regression are used for variable selection and QSPkR models development. The QSPkRs are evaluated by internal and external validation procedures.Results: A robust, significant and predictive QSPkR with explained variance r2 0.768, cross-validated q2LOO-CV 0.731,and geometric mean fold error of prediction (GMFEP) 1.79 is generated, which is able to predict the extent of PPB for 67.6% of the drugs in the dataset within the 2-fold error of experimental values. A simple empiric rule is proposed for distinguishing between drugs with different binding affinity, which allowed correct classification of 78% of the high binders and 87.5% of the low binders.Conclusions: PPB of neutral drugs is favored by lipophilicity, dipole moment, the presence of substituted aromatic and fused rings and a nine-member ring system, and is disfavored by the presence of aromatic N-atoms. Keywords: Plasma protein binding (PPB), Quantitative structure–pharmacokinetics relationship (QSPkR), In silico prediction, Human serum albumin (HSA), Alpha-1-acid glycoprotein (AGP)

Review of QSAR Models and Software Tools for predicting Biokinetic Properties

In the assessment of industrial chemicals, cosmetic ingredients, and active substances in pesticides and biocides, metabolites and degradates are rarely tested for their toxicologcal effects in mammals. In the interests of animal welfare and cost-effectiveness, alternatives to animal testing are needed in the evaluation of these types of chemicals. In this report we review the current status of various types of in silico estimation methods for Absorption, Distribution, Metabolism and Excretion (ADME) properties, which are often important in discriminating between the toxicological profiles of parent compounds and their metabolites/degradation products. The review was performed in a broad sense, with emphasis on QSARs and rule-based approaches and their applicability to estimation of oral bioavailability, human intestinal absorption, blood-brain barrier penetration, plasma protein binding, metabolism and. This revealed a vast and rapidly growing literature and a range of software tools. While it is difficult to give firm conclusions on the applicability of such tools, it is clear that many have been developed with pharmaceutical applications in mind, and as such may not be applicable to other types of chemicals (this would require further research investigation). On the other hand, a range of predictive methodologies have been explored and found promising, so there is merit in pursuing their applicability in the assessment of other types of chemicals and products. Many of the software tools are not transparent in terms of their predictive algorithms or underlying datasets. However, the literature identifies a set of commonly used descriptors that have been found useful in ADME prediction, so further research and model development activities could be based on such studies.JRC.DG.I.6-Systems toxicolog

CD, UV, and In Silico Insights on the Effect of 1,3-Bis(1′-uracilyl)-2-propanone on Serum Albumin Structure

1,3-diaryl-2-propanone derivatives are synthetic compounds used as building blocks for the realization not only of antimicrobial drugs but also of new nanomaterials thanks to their ability to self-assemble in solution and interact with nucleopeptides. However, their ability to interact with proteins is a scarcely investigated theme considering the therapeutic importance that 1,3-diaryl-2-propanones could have in the modulation of protein-driven processes. Within this scope, we investigated the protein binding ability of 1,3-bis(1'-uracilyl)-2-propanone, which was previously synthesized in our laboratory utilizing a Dakin-West reaction and herein indicated as U2O, using bovine serum albumin (BSA) as the model protein. Through circular dichroism (CD) and UV spectroscopy, we demonstrated that the compound, but not the similar thymine derivative T2O, was able to alter the secondary structure of the serum albumin leading to significant consequences in terms of BSA structure with respect to the unbound protein (Δβ-turn + Δβ-sheet = +23.6%, Δα = -16.7%) as revealed in our CD binding studies. Moreover, molecular docking studies suggested that U2O is preferentially housed in the domain IIIB of the protein, and its affinity for the albumin is higher than that of the reference ligand HA 14-1 (HDOCK score (top 1-3 poses): -157.11 ± 1.38 (U2O); -129.80 ± 6.92 (HA 14-1); binding energy: -7.6 kcal/mol (U2O); -5.9 kcal/mol (HA 14-1)) and T2O (HDOCK score (top 1-3 poses): -149.93 ± 2.35; binding energy: -7.0 kcal/mol). Overall, the above findings suggest the ability of 1,3-bis(1'-uracilyl)-2-propanone to bind serum albumins and the observed reduction of the α-helix structure with the concomitant increase in the β-structure are consistent with a partial protein destabilization due to the interaction with U2O

A biophysical insight of Camptothecin biodistribution: towards a molecular understanding of its pharmacokinetic issues

Camptothecin (CPT) is a potent anticancer drug, and its putative oral administration is envisioned although difficult due to physiological barriers that must be overcome. A comprehensive biophysical analysis of CPT interaction with biointerface models can be used to predict some pharmacokinetic issues after oral administration of this or other drugs. To that end, different models were used to mimic the phospholipid composition of normal, cancer, and blood–brain barrier endothelial cell membranes. The logD values obtained indicate that the drug is well distributed across membranes. CPT-membrane interaction studies also confirm the drug’s location at the membrane cooperative and interfacial regions. The drug can also permeate membranes at more ordered phases by altering phospholipid packing. The similar logD values obtained in membrane models mimicking cancer or normal cells imply that CPT has limited selectivity to its target. Furthermore, CPT binds strongly to serum albumin, leaving only 8.05% of free drug available to be distributed to the tissues. The strong interaction with plasma proteins, allied to the large distribution (VDSS = 5.75 ± 0.932 L·Kg−1) and tendency to bioaccumulate in off-target tissues, were predicted to be pharmacokinetic issues of CPT, implying the need to develop drug delivery systems to improve its biodistribution.This work was supported by Fundação para a Ciência e Tecnologia (FCT) in the framework of the Strategic Funding [UID/FIS/04650/2019], and by the project CONCERT [POCI-01- 0145-FEDER-032651 and PTDC/NAN-MAT/326512017], co-financed by the European Regional Development Fund (ERDF), through COMPETE 2020, under Portugal 2020, and FCT I.P. The authors thank Elettra Sincrotone and Sigrid Bernstorff, Trieste, Italy, for beam time and support through the project 20155321. Marlene Lúcio thanks FCT and ERDF for doctoral position [CTTI-150/18-CF (1)] in the ambit of the project CONCERT. Andreia Almeida (SFRH/BD/118721/2016) and Eduarda Fernandes (SFRH/BD/147938/2019) grants are supported by FCT, POPH and FEDER/COMPETE

In silico & In vitro study to estimate Plasma Protein Binding of anti-parasitic compounds for Sleeping sickness (Human African trypanosomiasis)

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a disease caused by a group of parasites called Trypanosoma brucei (Tb). The two main types causing HAT are T. brucei gambiense and T. brucei rhodesiense. T. brucei gambiense is the most common form of HAT, accounting for ninety seven percent of all reported cases of sleeping sickness. According to WHO, HAT is endemic in 36 sub-Saharan African countries. The disease can lead to death during the second stage if left untreated. Several drugs have been developed for the first stage such as pentamidine and suramin, and for the second stage such as melarsoprol, nifurtimox-eflornithine combination therapy (NECT). In 2019, fexinidazole was introduced as an oral treatment for the first stage and non-severe second stage of HAT. Several antiparasitic compounds prepared by our collaborator’s research group at the University of Graz, Austria showed varying levels of activity against Tb in vitro, whereas the compounds had only a moderate in vivo effect if at all. The suggested reason for the poor in vivo activities is that the compounds may bind tightly to plasma proteins, or they are metabolized before reaching the target sites for therapeutic effect. The prediction of plasma protein binding is of paramount importance in the pharmacokinetics characterization of drugs, as it causes significant changes in volume of distribution, clearance and drug half-life. Human serum albumin (HSA), an abundant plasma protein, can bind a remarkable variety of drugs impacting their delivery and efficacy and ultimately altering the drug’s pharmacokinetic and pharmacodynamic properties. In this current investigation, the overall aim was to investigate whether a strong HSA binding could be a probable reason for the poor in vivo activity of the provided antiparasitic compounds. The interaction of the antiparasitic compounds with HSA was studied computationally by docking them in the HSA drug binding site I and II. The compounds with the highest docking score were additionally studied using molecular dynamics simulations to evaluate the stability of the binding interactions. Moreover, the HSA binding affinity of the compounds was estimated by calculating the binding free energies using the MM-GBSA approach. In addition, experimental HSA binding studies using Microscale thermophoresis (MST) were conducted for some of the compounds. The results of the in silico studies suggest that majority of the investigated compounds may bind to HSA with varying affinity whereas a few of them did not show favorable binding interactions with HSA. Further, none of the compounds studied in vitro by MST showed HSA binding. In sum, plasma protein binding may be the reason for the in vivo inactivity for some of the investigated antiparasitic compounds

In vitro dosimetry of selected compounds and in vitro to in vivo extrapolation (IVIVE) for New Approach Methods (NAM) in toxicology

Potential hazard and risks of substances, pesticides and pharmaceuticals are traditionally addressed with animal experiments, for which numerous standardized, recognized and harmonized methods are available. In accordance with the paradigm of the 21st century concerning toxicological risk assessment and the 3R principle (“Reduction, Replacement, Refinement”), i.e. replacing animal experiments, in vitro and in silico based methods are increasingly being used to assess concentration-effect relationships. To derive risk assessments from results of in vitro methods, relevant human and animal doses should be extrapolated from concentrations in cell-based assays (in vitro-in vivo extrapolation, IVIVE). Usually, the effective concentration in vitro is based on a pre-defined amount of substance per unit cell culture volume, the so-called nominal concentration. However, factors such as binding to proteins in the cell culture medium, to adsorption to the cell culture vessel, and processes such as volatilization and enzymatic degradation contribute to the reduction of the concentration of a test substance in an in vitro test system. This hampers the extrapolation to relevant in vivo doses. The actual concentration of test substances in in vitro systems was examined in this work. For this purpose, analytical methods were developed, validated and used to determine actual in vitro concentrations of twelve test substances. Finally, the obtained effective in vitro concentrations were used to assess the endocrine disrupting potential of substances based on in vitro study results. The work on determining actual in vitro concentrations was divided into three parts: (1) Binding to proteins, which depends on the amount of protein in the medium, has a major influence on the unbound concentration of a substance in an in vitro test system. Likewise, substances bin to proteins, such as albumin, in blood plasma in vivo. Since the free fraction (fu) of a substance might be responsible for an effect, the fraction bound to proteins of the test substances in human plasma was determined. Applied methods for the investigation of protein binding are the rapid equilibrium dialysis (RED), ultrafiltration (UF) and ultracentrifugation (UC), which were checked for repeatability, accuracy and robustness in this work. In comparison to published literature data, more reliable fu ten test substances were obtained using RED with recovery values of 70 – 130 %. Physico-chemical properties of the substances, e.g. the octanol-water partition coefficient, are significant when selecting the appropriate separation method. The methods UF and UC are also recommended for polar substances (fu >70 % and logPow < 2) since nonspecific binding to devices are not significant, while the protein binding of lipophilic substances (logPow of 3.6 - 6.84) should be rather determined using RED based on comparable derived fu with literature data and suitable recovery values (68.2 – 118.1 %). Recoveries below 50 % and higher fu in comparison to published reference data were derived for moderately lipophilic to lipophilic substances when using UC of UF. (2) Methods for the quantification of test substances in the culture medium and cell lysates and as well as different sample preparations for the cell lysates were developed and the impact of sampling preparation on the measured cellular concentrations evaluated. A diffusion-based model for predicting the concentration in the compartments of the in vitro test system was used and the experimental data utilized to evaluate the model. Based on the results obtained from the protein binding studies, analytical concentrations of the test substances at 6, 24 and 48 h of incubation were corrected for the experimentally determined free fraction. The experiment was carried out with the Balb/c 3T3 cell line assuming that the cellular uptake of substances is based on diffusion due to the low transporter expression in this cell line. With regards to the mass balances and the measured data, minor effects on the concentration of the test substances with little or no protein binding in the culture medium were determined. On the other hand, substances with a defined mechanism of action and particular cellular targets or high octanol-water partition coefficient (TAM) showed deviations from the nominal concentration or analytically measurable concentration in the medium and thus resulted in a shift of the equilibrium towards the cell compartment. Cellular concentrations are up to 2 – 274 times higher than the nominal concentration and thus confirmed the discrepancies between medium and cell compartment. Computational and experimental data on the total and free concentration of the test substances in the culture medium agreed for eleven substances for which accordance below a factor of 2 were observed. In contrast, up to 4-fold higher concentrations were predicted for the Balb/c 3T3 cells. Discrepancies could be explained by the lack of involvement of target compartments such as cell organelles where substance specific mechanisms may be triggered (e.g. lysosomes as target organelle for tamoxifen), metabolic activation, transporter-mediated uptake and the degree of ionization of the molecules in the model. The model can be expanded by including these parameters in future work and needs to be validated again. The simple model can be used as a first screening method to assess the behavior of a substance in diffusion-based test systems. (3) Finally the approaches from previous work, the investigation on plasma protein binding and in vitro dosimetry, were combined with a reverse dosimetry approach and applied to in vitro effect concentrations in YES/YAS and steroidogenesis assay to determine oral doses in rats with regards to endocrine effects. For this purpose, concentrations of seven test substances (APAP, BPA, CAF, FEN, FLU, GEN, KET) were quantified in culture media and yeasts and human H295R adenocarcinoma cells using validated analytical methods. These serve as the POD for a QIVIVE based on physiologically based toxicokinetic model (PBTK model) to calculate an external, oral in vitro dose. This extrapolated in vivo does was and to compare the data obtained with published results on in vivo doses based on nominal in vitro concentrations. Increasing concentrations in cells has been found for for 4/7 test substances (BPA, FEN, FLU, GEN) which are known to have an affinity to the estrogenic/androgenic receptor or interfere with steroidogenesis respectively. An equilibrium between the medium and cell compartments was observed in the negative controls (APAP and CAF) which are known to not induce effects in the YES-/YAS- and steroidogenesis assay. Using cellular concentration, oral doses for 6/7 compounds were correctly calculated within a factor of 10, providing results with higher correlation to in vivo data than the estimated LOEL based on total and medium concentrations. However, the study highlights the analytical difficulties that arise when determining cellular concentrations over multiple time points (3, 6 and 12 h). Accordingly, no statements could be made on the actual substance uptake over time in the YES-/YAS-assay (APAP, BPA, CAF, FLU, GEN). Nominal as well as total concentrations of substances can serve as POD if there is no affinity to cellular targets, low protein binding (less than 10%) and no metabolic activation by the cell lines are present. Future studies may include other endocrine disruptors and promote the use of reverse dosimetry in relation to other toxicological endpoints

The Mycobacterium tuberculosis Drugome and Its Polypharmacological Implications

We report a computational approach that integrates structural bioinformatics, molecular modelling and systems biology to construct a drug-target network on a structural proteome-wide scale. The approach has been applied to the genome of Mycobacterium tuberculosis (M.tb), the causative agent of one of today's most widely spread infectious diseases. The resulting drug-target interaction network for all structurally characterized approved drugs bound to putative M.tb receptors, we refer to as the ‘TB-drugome’. The TB-drugome reveals that approximately one-third of the drugs examined have the potential to be repositioned to treat tuberculosis and that many currently unexploited M.tb receptors may be chemically druggable and could serve as novel anti-tubercular targets. Furthermore, a detailed analysis of the TB-drugome has shed new light on the controversial issues surrounding drug-target networks [1]–[3]. Indeed, our results support the idea that drug-target networks are inherently modular, and further that any observed randomness is mainly caused by biased target coverage. The TB-drugome (http://funsite.sdsc.edu/drugome/TB) has the potential to be a valuable resource in the development of safe and efficient anti-tubercular drugs. More generally the methodology may be applied to other pathogens of interest with results improving as more of their structural proteomes are determined through the continued efforts of structural biology/genomics

Biological Evaluation and Molecular Docking Studies of Benzalkonium Ibuprofenate

The third-generation ionic liquids (ILs), which are being used to produce double active pharmaceutical ingredients (d-APIs) with tunable biological activity along with novel performance, enhancement, and delivery options, have been revolutionizing the area of drug discovery since the past few decades. Herein we report the in vitro antibacterial and anti-inflammatory activity of benzalkonium ibuprofenate (BaIb) that are being used as in-house d-API, with a particular focus on its interaction with respective protein target through molecular docking study. The evaluation of the biological activity of BaIb with the antibacterial and anti-inflammatory target at the molecular level revealed that the synthesized BaIb could be designed as a potential double active drug since it retains the antibacterial and anti-inflammatory activity of its parent drugs, benzalkonium chloride (BaCl) and sodium ibuprofenate (NaIb), respectively

Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays