Physiologically relevant partition coefficients between an aqueous phase (plasma), lipids (cell membrane), and proteins are lacking for many per- and polyfluoroalkyl substances (PFAS). Biomimetic chromatography utilizes immobilized artificial membrane (IAM), human serum albumin (HSA), and alpha-1-glycoprotein (AGP) stationary phases to determine partition coefficients. A rapid-gradient biomimetic approach was used to determine partition coefficients for 81 target PFAS and 37 suspect PFAS in a 3M aqueous film forming foam. PFAS were categorized as acids, bases, or neutrals and used to interpret lipophilic contributions to binding on IAM, HSA, and AGP columns. Partition coefficients on the IAM membrane (log KIAM: 1.4 to 7.9; median 3.4) and AGP protein (log kAGP: -1.9 to 0.72; median 0.31) increased linearly with increasing fluorinated chain length, while those for HSA protein (log KHSA: 0.45 to 5.1; median 4.3) reached a plateau at a specific chain length (∼C6-C8). Zwitterionic PFAS at pH 7.4 exhibited lower affinities for both the IAM membrane and HSA protein, which are likely due to the smaller fraction of the charged species at pH 7.4. The partition coefficients (CHI log D [chromatographic log D derived from the Chromatographic Hydrophobicity Index scaled to the octanol/water partition coefficient, log D]), log KIAM, log KHSA, and log kAGP) expand our understanding of how PFAS behave in the body and provide a foundation for future toxicokinetic modeling.The rapid-gradient approach offers a novel method for measuring partition coefficients for PFAS in complex mixtures.

Blood concentration thresholds associated with therapeutic, toxic, and lethal effects reflect distribution-related processes but also pharmacodynamic, clinical, and forensic factors. This study examined whether biomimetic chromatographic descriptors provide information distinct from conventional physicochemical descriptors, or primarily serve as integrative proxies for the same underlying descriptor space. A curated dataset of 92 drugs was modeled using biomimetic descriptors, including CHIIAM, log kHSA, and log kAGP, alongside conventional variables such as log P, log D₇.₄, molecular weight, hydrogen-bonding, polarity, and ionization descriptors. CHIIAM was the most informative single descriptor across all endpoints (R² ≈ 0.25-0.32), indicating that membrane-affinity-related properties contribute to systemic concentration thresholds. Multivariate models improved performance substantially (R² ≈ 0.62-0.73; Q²LOO ≈ 0.57-0.70), but biomimetic and conventional models showed nearly equivalent resampling performance (Q²pred ≈ 0.44-0.59). Nested model analysis showed that adding CHIIAM improved incomplete conventional models (ΔR² up to 0.28, p < 0.001), whereas adding conventional descriptors to CHIIAM-based models provided limited additional benefit. These findings indicate that CHIIAM functions as a compact experimental descriptor integrating lipophilicity, ionization, polarity, and molecular-size effects that conventional models reconstruct through multiple calculated variables. Robustness was supported by repeated validation, Y-randomization, and applicability-domain analysis, although predictive performance remained moderate and internally validated. Biomimetic chromatography offers an experimentally grounded low-dimensional representation of distribution-related physicochemical space, whereas conventional descriptors provide greater mechanistic decomposability. These results support the flexible integration of biomimetic and calculated descriptors for early-stage drug distribution and toxicological modeling, while emphasizing their use for descriptor interpretation and comparative screening rather than for direct clinical or forensic prediction.

Background and purpose: Organic UV filters are commonly used in sunscreen and cosmetic formulations to protect against harmful UV radiation. However, concerns have emerged over their potential toxic effects on aquatic organisms. This study aims to investigate the acute aquatic toxicity of 13 organic UV filters and determine whether phospholipid binding, measured through biomimetic chromatographic methods, is a better predictor of toxicity than the traditionally used octanol-water partition coefficient (log P). Experimental approach: The chromatographic retention of the 13 UV filters was measured on an immobilized artificial membrane (IAM) stationary phase to assess phospholipid binding. These measurements were then applied to previously established predictive models, originally developed for pharmaceutical compounds, to estimate acute aquatic toxicity endpoints of 48-hour LC50 for fish and the 48-hour EC50 (immobilization) for Daphnia magna. Key results: Phospholipid binding was found to be a more reliable predictor of the acute aquatic toxicity of UV filters compared to log P. The toxicity was primarily driven by lipophilicity and charge, with negatively charged compounds exhibiting lower toxicity. Conclusion: The study demonstrates that phospholipid binding is a better descriptor of UV filter toxicity than log P, providing a more accurate method for predicting the environmental risk of these compounds. This insight can guide the development of more environmentally friendly sunscreens by reducing the use of highly lipophilic and positively charged compounds, thus lowering their aquatic toxicity.

Biomimetic chromatography is the name of the High Performance Liquid Chromatography (HPLC) methods that apply stationary phases containing proteins and phospholipids that can mimic the biological environment where drug molecules distribute. The applied mobile phases are aqueous organic with a pH of 7.4 to imitate physiological conditions that would be encountered in the human body. The calibrated retention of molecules on biomimetic stationary phases reveals a compound's affinity to proteins and phospholipids, which can be used to model the biological and environmental fate of molecules. This technology, when standardised, enables the prediction of in vivo partition and distribution behaviour of compounds and aids the selection of the best compounds for further studies to become a drug molecule. Applying biomimetic chromatographic measurements helps reduce the number of animal experiments during the drug discovery process. New biomimetic stationary phases, such as sphingomyelin and phosphatidylethanolamine, widen the application to the modelling of blood-brain barrier distribution and lung tissue binding. Recently, the measured properties have also been used to predict toxicity, such as phospholipidosis and cardiotoxicity. The aquatic toxicity of drugs and pesticides can be predicted using biomimetic chromatographic data. Biomimetic chromatographic separation methods may also be extended in the future to predict protein and receptor binding kinetics. The development of new biomimetic stationary phases and new prediction models will further accelerate the widespread application of this analytical method.

The potential of biomimetic chromatography to predict ecotoxicological endpoints of pharmaceutical compounds was investigated. For this purpose, a data set of previously and newly measured chromatographic retention data for 36 structurally diverse drugs was used. Standardized retention times were measured on the immobilized artificial membrane, human serum albumin, and alpha-1-acid glycoprotein stationary phases. As ecotoxicological endpoints, half-maximal lethal concentration values of fish and half-maximal effective concentration (immobilization) values of a water flea (Daphnia magna spp.) determined with a two-day static method were considered. Ecotoxicity values correlated with octanol-water partitioning and the positive charge of compounds contributed even more to the toxicity. Models based on membrane partition exhibited the best statistics and predictive performance, attributed to lipophilicity and membrane electrostatic interactions. Alpha-1-acid glycoprotein binding led to satisfactory models, owing to its function as a binder of neutral and basic lipophilic compounds. Albumin binding, however, did not result in sound models, as it is governed by lipophilicity and the negative charge of compounds, contrary to the mechanism of toxicity. Both membrane and alpha-1-acid glycoprotein models were superior statistically from those derived from the octanol-water system. Overall, membrane and alpha-1-acid glycoprotein retention can be suggested as promising indices to assess the ecotoxicological risk of drugs.

Inhibitors of Kelch-like ECH-associated protein 1 (Keap1) increase the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) by stalling its ubiquitination and degradation. This enhances the expression of genes encoding proteins involved in drug detoxification, redox homeostasis, and mitochondrial function. Nrf2 activation offers a potential therapeutic approach for conditions including Alzheimer's and Parkinson's diseases, vascular inflammation, and chronic obstructive airway disease. Non-electrophilic Keap1-Nrf2 protein-protein interaction (PPI) inhibitors may have improved toxicity profiles and different pharmacological properties to cysteine-reactive electrophilic inhibitors. Here, we describe and characterize a series of phenyl bis-sulfonamide PPI inhibitors that bind to Keap1 at submicromolar concentrations. Structural studies reveal that the compounds bind to Keap1 in a distinct "peptidomimetic" conformation that resembles the Keap1-Nrf2 ETGE peptide complex. This is different to other small molecule Keap1-Nrf2 PPI inhibitors, including bicyclic aryl bis-sulfonamides, offering a starting point for new design approaches to Keap1 inhibitors.

The major causes of failure of drug discovery compounds in clinics are the lack of efficacy and toxicity. To reduce late-stage failures in the drug discovery process, it is essential to estimate early the probability of adverse effects and potential toxicity. Cardiotoxicity is one of the most often observed problems related to a compound's inhibition of the hERG channel responsible for the potassium cation flux. Biomimetic HPLC methods can be used for the early screening of a compound's lipophilicity, protein binding and phospholipid partition. Based on the published hERG pIC50 data of 90 marketed drugs and their measured biomimetic properties, a model has been developed to predict the hERG inhibition using the measured binding of compounds to alpha-1-acid-glycoprotein (AGP) and immobilised artificial membrane (IAM). A representative test set of 16 compounds was carefully selected. The training set, involving the remaining compounds, served to establish the linear model. The mechanistic model supports the hypothesis that compounds have to traverse the cell membrane and bind to the hERG ion channel to cause the inhibition. The AGP and the hERG ion channel show structural similarity, as both bind positively charged compounds with strong shape selectivity. In contrast, a good IAM partition is a prerequisite for cell membrane traversal. For reasons of comparison, a corresponding model was derived by replacing the measured biomimetic properties with calculated physicochemical properties. The model established with the measured biomimetic binding properties proved to be superior and can explain over 70% of the variance of the hERG pIC50 values.

Abstract This chapter describes the application of various separation methods for the measurements of physicochemical and biomimetic properties of early drug discovery compounds. The retention on reversed-phase stationary phases is proportional to the lipophilicity, while the retention using chemically bonded protein stationary phases and immobilized artificial membrane stationary phases is proportional to the protein and phospholipid binding of compounds. The retention in supercritical fluid chromatography can reveal the accessible polar surface area of the compounds, while the retention using orthogonal high-performance liquid chromatography systems can reveal the H-bond acidity, H-bond basicity and dipolarity/polarizability. These properties then can be used to build models to predict the in vivo behaviour of putative drug molecules such as the volume of distribution, drug efficiency, permeability, blood–brain barrier distribution, and so help the design of new drug molecules. Throughout this chapter, examples are given of the application of physicochemical and biomimetic measurement techniques with full and comprehensive explanations.

Thin-layer chromatography (TLC) will continue to play a basic role in determining the separation possibilities of chromatographic methods for the routine analysis of large numbers of samples or to analyze samples in cases where high-performance liquid chromatography has difficulties. TLC has the advantage that it does not require sophisticated and expensive analytical instrumentation. Several samples can be analyzed simultaneously and it provides the possibility of a wide variety of detection methods for the separated components either by using UV or chemical reactions resulting in coloured spots. Therefore, TLC procedures are important especially in resource-limited countries or laboratories. A number of further instrumental developments have been made to improve the sample application on the plates and in applying controlled and pressurized mobile-phase flow. The detection and further analysis of the separated components can be carried out using sophisticated instrumentation such as mass spectrometry and infrared spectroscopy as mentioned in this chapter.

Immobilized Artificial Membrane (IAM) chromatography columns have been used to model the in vivo distribution of drug discovery compounds. Regis Technologies Inc., the manufacturer, had to replace the silica support and consequently introduced a new IAM.PC.DD2 column that shows slightly different selectivity towards acidic and basic compounds. The application of the new IAM.PC.DD2 columns has been evaluated and the in vivo distribution models have been compared with the previous batches of columns. It was found that due to the improved endcapping of the silica, some of the positively charged drug molecules showed shorter retention than previously published. Therefore, the column system suitability data have been updated. However, these differences do not significantly affect the previously published models for the volume of distribution, brain tissue binding and drug efficiency. Therefore, the published models can be used with the new IAM.PC.DD2 columns.

Chloroquine and hydroxy-chloroquine already established as anti-malarial and lupus drugs have recently gained renewed attention in the fight against the Covid-19 pandemic. Bio-mimetic HPLC methods have been used to measure the protein and phospholipid binding of the racemic mixtures of the drugs. The tissue binding and volume of distribution of the enantiomers have been estimated. The enantiomers can be separated using Chiralpak AGP HPLC columns. From the α-1-acid-glycoprotein (AGP) binding, the lung tissue binding can be estimated for the enantiomers. The drugs have a large volume of distribution, showed strong and stereoselective glycoprotein binding, medium-strong phospholipid-binding indicating only moderate phospholipidotic potential, hERG inhibition and promiscuous binding. The drug efficiency of the compounds was estimated to be greater than 2 % which indicates a high level of free biophase concentration relative to dose. The biomimetic properties of the compounds support the well-known tolerability of the drugs.

Three promising antibacterial peptides were studied with regard to their ability to inhibit the growth and kill the cells of clinical strains of Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. The multifunctional gramicidin S (GS) was the most potent, compared to the membranotropic temporin L (TL), being more effective than the innate-defence regulator IDR-1018 (IDR). These activities, compared across 16 strains as minimal bactericidal and minimal inhibitory concentrations (MIC), are independent of bacterial resistance pattern, phenotype variations and/or biofilm-forming potency. For S. aureus strains, complete killing is accomplished by all peptides at 5 × MIC. For E. faecalis strains, only GS exhibits a rapid bactericidal effect at 5 × MIC, while TL and IDR require higher concentrations. The biofilm-preventing activities of all peptides against the six strains with the largest biofilm biomass were compared. GS demonstrates the lowest minimal biofilm inhibiting concentrations, whereas TL and IDR are consistently less effective. In mature biofilms, only GS completely kills the cells of all studied strains. We compare the physicochemical properties, membranolytic activities, model pharmacokinetics and eukaryotic toxicities of the peptides and explain the bactericidal, antipersister and antibiofilm activities of GS by its elevated stability, pronounced cell-penetration ability and effective utilization of multiple modes of antibacterial action.

Characterizing the properties of large numbers of compounds and estimating their potential absorption, distribution, metabolism and elimination properties are important early stages in the process of drug discovery and help to reduce later stage attrition. The chromatographic separation principles using stationary phases that contain proteins and phospholipids are more suitable for compound characterization and estimation of the pharmacokinetic properties than the traditional octanol/water partition coefficient. This technology, when standardized, enables the prediction of in vivo behavior and the selection of compounds with the best potential, thus reducing the number of animal experiments. Chromatography may be involved more widely in the future to measure kinetic aspects of compounds’ binding to proteins and receptors which would enable designing compounds that require a lower frequency of doses and have more predictable pharmacokinetic profiles.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pK(a), CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.

Amyotrophic lateral sclerosis (ALS) is an idiopathic, fatal neurodegenerative disease of the human motor system. Subunits of the 33-amino acid containing motorneurontrophic factor (MNTF) have been investigated and GM6 has been found as a potential peptide therapeutic for ALS. This linear peptide drug candidate has been characterized by HPLC based physicochemical and biomimetic measurements to estimate its in vivo distribution behavior and to estimate its cell penetration and brain to plasma concentration ratio. The free tissue concentration vs time profile has been estimated using the measured physicochemical and biomimetic properties of the intact GM6 molecules and its microsomal stability. The in vitro and in vivo measurements supported the estimated in vivo distribution behavior of GM6.

The drug discovery process can be accelerated by chromatographic profiling of analogs by measuring their nonspecific binding to proteins and lipids and then by modeling in vivo distribution. A balanced potency and chromatographically determined membrane and protein binding ensure the selection of compounds with the highest probability to show the desired in vivo distribution behavior for efficacy and reduced toxicity. The first part of the article will discuss the high performance liquid chromatography (HPLC)-based measurements of lipophilicity and biomimetic properties, and the second part will discuss the models derived from the measured data of known drug molecules and drug discovery compounds.

The biomimetic gradient retention time measurements on C18, immobilized artificial membrane (IAM), human serum albumin (HSA), and acid-glycoprotein (AGP) stationary phases can be used to characterize compounds partitioning into phospholipids and proteins. The data obtained can then be used in equations to estimate the in vivo plasma-tissue distribution of the compounds measured. The plasma protein binding, brain tissue binding, and in vivo drug efficiency can also be calculated using the biomimetic chromatographic data.