In vitro measurement of drug efficiency index to aid early lead optimization.

The concepts of drug efficiency (D(eff) ) and Drug Efficiency Index (DEI) have been recently introduced as useful parameters to optimize the absorption, distribution, metabolism, elimination/excretion, and toxicity properties and in vivo efficacy potential of molecules during lead optimization and at pre-clinical stages. The available free drug concentration relative to dose depends on the compound's bioavailability, clearance, and the nonspecific binding to proteins and phospholipids. In this paper, we have demonstrated, using the data of over 115 known drug molecules, that the nonspecific binding can be determined in vitro very efficiently using biomimetic high-performance liquid chromatography measurements. DEI can therefore be estimated from in vitro measurements. The data show that high in vitro DEI values can be associated with lower efficacious dose. A strategy is described of how to use the DEI parameter during early lead optimization. An example is given to highlight the advantages of optimizing on DEI value rather than on potency alone. In order to facilitate the in silico compound design, correlation between in vitro DEI and in silico ligand efficiency parameters such as ligand lipophilicity efficiency has been revealed, suggesting the potential use of these efficiency-related parameters across lead optimization.

Physicochemical profile of macrolides and their comparison with small molecules.

Macrolides are stereospecific macrolactones of high molecular weights. Herein, 600 mostly semisynthetic macrolides are compared with 50,000 small non-macrolide synthetic molecules in terms of measured physicochemical properties in order to assess the drug-likeness and developability chances of macrolides. The pre-selected set of diverse macrolides is comprised mostly of derivatives of clarithromycin and azithromycin cores. Lipophilicity (CHI logD), affinity for immobilized artificial membranes (CHI IAM), human serum albumin (HSA) and α(1)-acid glycoprotein (AGP) plasma protein bindings (PPB), DMSO precipitative solubility as well as artificial membrane permeability (AMP) have been determined by high-throughput screening methods. It has been found that macrolides and small molecules have similar lipophilicity profiles, though macrolides show weaker PPB and have better solubility than small discovery molecules. However, macrolides are poorly permeable and have high affinity for immobilized artificial membranes signifying their strong interaction with biological phospholipids. In order to retain the drug-like profile, the design of novel macrolide molecules should be focused on optimisation of macrolide cores, that is macrolactone moiety with sugars and other small substituents avoiding large substituents and flexible linkers such as in conjugate derivatives.

Estimating unbound volume of distribution and tissue binding by in vitro HPLC-based human serum albumin and immobilised artificial membrane-binding measurements.

The in vivo unbound volume of distribution (V(du)) can be used to estimate the free steady-state plasma concentration with a given dose of a drug administered intravenously. We have demonstrated that the calibrated HPLC retention times obtained on biomimetic stationary phases, such as immobilised human serum albumin and phosphatidyl-choline, can be used to estimate compounds' in vivo behaviour. The mechanistic models are based on the assumption that the sum of the albumin and phospholipid binding has the most significant impact on reducing compounds' free concentration both in plasma and in tissues. The model equations were obtained using the literature human volume of distribution and fraction unbound in plasma values of 135 known drug molecules and have been tested on a further 300 in-house compounds. The model can be used to design compounds with low V(du) values and high fraction unbound in tissues which will minimise the required dose to achieve the efficacious free concentration at the target organ (excluding possible active transport processes).

Estimation of volume of distribution in humans from high throughput HPLC-based measurements of human serum albumin binding and immobilized artificial membrane partitioning.

The volume of distribution (VD) in humans of 179 known drug molecules (acids, bases, and neutrals) has been modeled using two biomimetic-binding measurements. The phospholipid binding (log K (IAM)) and the plasma protein binding (log K (HSA)) have been calculated from gradient HPLC retention times on immobilized artificial membrane (IAM) and on human serum albumin (HSA) columns, respectively. The log VD values showed good correlation with the compounds' relative binding to IAM and HSA as follows: log VD=0.44 log K (IAM)-0.22 log K (HSA)-0.66; n=179, r2=0.76, s=0.33, and F=272. It was also observed that positively charged molecules bind relatively more to IAM, while negatively charged ones bind more to HSA, in line with the empirical observation that bases tend to have a larger volume of distribution than acids. These results suggest that with the help of these two simple high throughput HPLC-based biomimetic binding measurements an important in vivo drug disposition property can be estimated for use in early drug discovery.

Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilized artificial membrane lipophilicity.

A fast gradient HPLC method (cycle time 15 min) has been developed to determine Human Serum Albumin (HSA) binding of discovery compounds using chemically bonded protein stationary phases. The HSA binding values were derived from the gradient retention times that were converted to the logarithm of the equilibrium constants (logK HSA) using data from a calibration set of molecules. The method has been validated using literature plasma protein binding data of 68 known drug molecules. The method is fully automated, and has been used for lead optimization in more than 20 company projects. The HSA binding data obtained for more than 4000 compounds were suitable to set up global and project specific quantitative structure binding relationships that helped compound design in early drug discovery. The obtained HSA binding of known drug molecules were compared to the Immobilized Artificial Membrane binding data (CHI IAM) obtained by our previously described HPLC-based method. The solvation equation approach has been used to characterize the normal binding ability of HSA, and this relationship shows that compound lipophilicity is a significant factor. It was found that the selectivity of the "baseline" lipophilicity governing HSA binding, membrane interaction, and octanol/water partition are very similar. However, the effect of the presence of positive or negative charges have very different effects. It was found that negatively charged compounds bind more strongly to HSA than it would be expected from the lipophilicity of the ionized species at pH 7.4. Several compounds showed stronger HSA binding than can be expected from their lipophilicity alone, and comparison between predicted and experimental binding affinity allows the identification of compounds that have good complementarities with any of the known binding sites.