In Vitro ADME, Physicochemical and General Biochemical Tests

Life Chemicals offers a highly efficient and solid framework of drug discovery-related services to companies and academic institutions working in the field of pharmaceutical and biotech research.

Available among our products is a variety of in vitro ADMET, physicochemical and biochemical tests of product candidates, hit and lead compounds to facilitate drug development projects, providing an exceptionally reliable and profound analysis within the most convenient timeline.

Please, contact us at orders@lifechemicals.com for any additional information and price quotations.

Detailed Method Description

High-resolution Mass Spectrometry Services

The Agilent 6545 Q-TOF is a high-resolution mass spectrometer suitable for accurate mass measurement with a molecular formula determination, as well as MS/MS studies. It is capable of ionization using Jet Stream ion sources with conventional collision-induced dissociation (CID). SWARM autotunes technology provides substantial sensitivity, increasing up to 5x fold for small molecules' detection, as well as helping to preserve fragile compounds for detection. Samples can be introduced directly via syringe infusion, or via Agilent 1260 UHPLC System. For those compounds which have a limited ability of ionization, the Multimode ion source is also available.

Proteomics and metabolomics services are also available upon request.

For more information, please check the following:

• Agilent 6545 Q-TOF LC/MS - presentation video on YouTube
• Agilent 6545 Q-TOF LC/MS System (pdf brochure)
• Six ways that Agilent Q-TOF technology will help you achieve excellent spectral quality (pdf brochure)

Solubility: HT Thermodynamic and Kinetic Methods

Solubility is one of the most essential physicochemical properties of drug candidates, and its measurement is an important component in the in vitro profiling of drug-like properties. It can be defined as the maximum amount of a substance that will dissolve in a given amount of solvent at a specific temperature. As far as medicines are concerned, poor solubility is detrimental to absorption after oral administration and can mask compound activity in bioassays in various ways. Measuring solubility in the early-stage drug discovery process can identify potentially ambiguous activity data, such as false negatives due to poor aqueous solubility during functional assays, thus, improving the overall efficacy of activity screening and hit identification. In addition, compounds with low aqueous solubility tend to be highly bound to plasma proteins with poor tissue distribution and increased potential for CYP enzyme inhibition.

Being revealed during earlier discovery stages, this property provides valuable information for better interpretation of screening results and helps design new molecules for the further drug development process. With the growing number of compounds as potential drug candidates, solubility assays for high throughput screening to enable rapid evaluation of many compounds are becoming increasingly important.

To best meet the research needs of our customers Life Chemicals carries out solubility measurements by both thermodynamic and kinetic methods.

Solubility by HT thermodynamic method: highlights

• The high-throughput thermodynamic method for solubility measurement enables rapid and accurate evaluation of numerous compounds

• Solubility is determined by dispensing a solid compound into a solvent (DMSO and PBS) with further HPLC quantification

• More often the results of thermodynamic solubility determinations tend to refer to a crystalline phase 

• Resulting concentrations are dependent on compound purity, ionization states, stability in solution, and other factors, such as solution temperature and pH

• The experimental protocol is available on request

Solubility by HT kinetic method: highlights

• The high-throughput kinetic method for solubility measurement uses only a few samples and allows its rapid determination
• The non-equilibrium solubility method can be applied during early drug discovery to evaluate new chemical entities, as well as for further lead optimization
• The purpose of the measurements is to identify compounds that do not possess a good kinetic solubility even in aqueous buffer containing DMSO, to guide modification of structures to improve solubility, and also to guide formulation selection for animal dosing
• Kinetic solubility data vary with the conditions of the solution: small changes in pH, organic solvent, ionic strength, ions in solution, co-solutes, incubation time, and temperature can result in significant changes in the solubility of a compound

LogP Determination

N-octanol-water partition coefficient (or simplified LogP) of a compound is a key parameter used in drug design and regulatory compliance, chemical manufacturing industry (environmental impact compliance), and the environmental field (the environmental fate of toxic substances). It has been widely used for:

• Design of drugs and pharmaceuticals
• Prediction and correlation of bioconcentration and soil and sediment sorption of organic pollutants
• Research on medicinal chemicals
• Modeling of the environmental fate of organic chemicals
• Toxicology of substances

Despite the possibility to calculate this parameter, the precision of obtained data might be different from experimental, causing an increasing discrepancy in biological tests. Therefore, experimental determination of LogP/LogD is the essential basic step prior to "hits" evaluation.

The Shake Flask LogP/LogD method we offer is based on OECD Guidelines for the testing of chemicals, supplemented with the HPLC method for the octanol-water system.

LogP and LogD by hybrid HPLC/shake-flask method: highlights

• A classical and most reliable method of the logP determination
• The partition coefficient is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two-phase system consisting of two largely immiscible solvents
• Concentrations of compounds in each phase are quantified with the HPLC method
• A low amount of solid compound is required (further diluted in DMSO) to perform the test
• The experimental protocol is available on request

Plasma Protein Binding (PPB)

The effect of organic compound binding to blood plasma proteins (Plasma Protein Binding (PPB)) is of particular interest in ADMET studies, since the bound compounds, according to the “free drug hypothesis”, cannot interact with their protein target. However, these interactions are also important for the creation of sustained-release dosage forms. The role of plasma protein binding is recognized as a major factor in the distribution and effectiveness of a potential drug.

Although immunoglobulins, lipoproteins, fibrinogen, and phospholipids contribute significantly to binding, and they can also greatly contribute to lowering free drug concentrations, it is generally accepted that mainly occurring is the binding with human serum albumin (HSA) which it binds mainly hydrophobic and negatively charged compounds, and alpha-1-acid glycoprotein (AGP or AAG) which it binds positively charged compounds.

Based on these proteins, Dr. Klara Valko has developed methods of bio-mimetic chromatography to evaluate PPB using exclusively HPLC. We offer PBB assessment using the modified methodology based on her previous work [1].

Plasma protein binding by bio-mimetic chromatography method: Highlights

• Bio-mimetic chromatography allows modeling a compound distribution in vivo and estimates its affinity with human non-specific binding components by using human proteins and phospholipid as biorelevant stationary phase
• The basic principle of the methodology is that the retention time of a compound (as a part of the mobile phases) passing through the HPLC column (containing three biorelevant stationary phases) is directly proportional to its affinity/dynamic equilibrium with the stationary phase
• Application of bio-mimetic chromatography reduces animal testing and late-stage attrition, lowers candidate selection cost, and allows early dose estimation
• We have modified the original methodology (proposed by Dr. K. Valko) to apply it for high-accuracy plasma protein binding (PPB) detection [1]. It has successfully been validated using experimental literature data for the percentage of PPB of known compounds
• The experimental protocol is available on request

Volume of Distribution (Log Vss)

The pharmacologically active compound is intended to interact with the target enzyme or receptor. However, only very small fractions of the administered drug achieve the goal in vivo. The distribution of the compound in the body depends on its affinity to many other components, such as proteins, nucleotides, and phospholipids. Since they are present in much larger volumes than the target enzyme or receptor, they significantly reduce the concentration of the active substance at the site of action. The distribution of the drug molecule in vivo depends on its properties. It may have higher local concentrations in certain tissues or organs.

Based on Dr. Klara Valko’s methods of bio-mimetic chromatography, we have developed a modified model for evaluation of the volume of distribution using exclusively HPLC [1].

Volume of distribution by bio-mimetic chromatography method: highlights

• Bio-mimetic chromatography allows modeling a compound distribution in vivo and estimates its affinity to human non-specific binding components by using human proteins and phospholipid as biorelevant stationary phases
• The basic principle of the methodology is that the retention time of a compound (as a part of the mobile phases) passing through the HPLC column (containing three biorelevant stationary phases) is directly proportional to its affinity/dynamic equilibrium with the stationary phase
• Application of bio-mimetic chromatography reduces animal testing and late-stage attrition, lowers candidate selection cost, and allows early dose estimation
• We have modified the original methodology (proposed by Dr. K. Valko) to apply it for the high-accuracy volume of distribution detection [1]. It has successfully been validated using experimental literature data for known compounds
• The experimental protocol is available on request

Unbound Volume of Distribution (Log Vdu)

Good in vitro efficacy and selectivity does not necessarily mean good in vivo efficacy due to poor absorption, high metabolic rate, or simply low free concentration in vivo. Binding to plasma proteins and tissue binding lower the free concentration of the drug molecule in vivo. To maintain the necessary effectiveness, it is required to raise the dose of the drug, which may increase the risk of side effects.

Since plasma proteins are present in high concentrations, even weak interactions can cause a slight decrease in the free concentration of the drug. The unbound volume of distribution (Vdu), which is defined as the dose/concentration of free plasma, gives a better estimate of the free drug available for interaction with the target in vivo than the binding of plasma protein per se.

Based on Dr. Klara Valko’s methods of bio-mimetic chromatography, we have developed a modified model for evaluation of the unbound volume of distribution using exclusively HPLC [1-2].

Unbound volume of distribution (log Vdu) by bio-mimetic chromatography method: highlights

• Bio-mimetic chromatography allows modeling a compound distribution in vivo and estimates its affinity to human non-specific binding components by using human proteins and phospholipid as biorelevant stationary phases
• The basic principle of the methodology is that the retention time of a compound (as a part of the mthe obile phases) passing through the HPLC column (containing three biorelevant stationary phases) is directly proportional to its affinity/dynamic equilibrium with the stationary phase
• Application of bio-mimetic chromatography reduces animal testing and late-stage attrition, lowers candidate selection cost, and allows early dose estimation
• We have modified the original methodology by Dr. K. Valko to apply it for the high-accuracy unbound volume of distribution detection [1-2]. It has successfully been validated using experimental literature data for known compounds
• The experimental protocol is available on request

Brain Tissue Binding

Estimating the unbound fraction of drugs in the brain has become essential for the evaluation and interpretation of the pharmacokinetics and pharmacodynamics of new central nervous system drug candidates. Several brain binding assays have been developed and some of them are widely applied in central nervous system (CNS) research. Many of the methods are similar to plasma protein binding (PPB) methods with slight modifications. Equilibrium dialysis with brain homogeneity to measure brain tissue binding is the gold standard method and is widely applied in drug discovery research and development.

At Life Chemicals, we offer our in-house developed modification of Dr. Klara Valko’s method of bio-mimetic chromatography to assess brain tissue binding, using exclusively HPLC [1].

Brain tissue binding by bio-mimetic chromatography method: highlights

• Bio-mimetic chromatography allows modeling a compound distribution in vivo and estimates its affinity to human non-specific binding components by using human proteins and phospholipid as biorelevant stationary phases
• The basic principle of the methodology is that the retention time of a compound (as a part of the mobile phases) passing through the HPLC column (containing three biorelevant stationary phases) is directly proportional to its affinity/dynamic equilibrium with the stationary phase
• Application of bio-mimetic chromatography reduces animal testing and late-stage attrition, lowers candidate selection cost, and allows early dose estimation
• We have modified the original methodology by K. Valko to apply it for high-accuracy brain tissue binding detection [1]. It has successfully been validated using experimental literature data for known compounds
• The experimental protocol is available on request

Lung Tissue Binding

The lungs are pharmacologically active organs, affecting the blood concentrations of drugs given intravenously. The lungs can take up, retain, metabolize and delay the release of many drugs and compounds.

Pulmonary surfactant is a mixture of lipids and proteins (approximately 90 % lipids and 10 % proteins), which is secreted by type II epithelial cells into the alveolar space. Its main function is to reduce surface tension at the air/liquid interface in the lungs. This is achieved by forming a surface film consisting of a monolayer that is enriched with dipalmitoylphosphatidylcholine (in all mammalian species, the surfactant contains approximately 80 % phosphatidylcholine) and two-layer lipid-protein structures that are closely related to it.

Studying and assessing the pharmacokinetic function of the lungs is very important in the drug development process. Based on Dr. Klara Valko’s methods of bio-mimetic chromatography, we have developed the modified model for the evaluation of lung tissue binding, using exclusively HPLC [1].

Lung tissue binding by bio-mimetic chromatography method: highlights

• Bio-mimetic chromatography allows modeling a compound distribution in vivo and estimates its affinity to human non-specific binding components by using human proteins and phospholipid as biorelevant stationary phases
• The basic principle of the methodology is that the retention time of a compound (as a part of the mobile phases) passing through the HPLC column (containing three biorelevant stationary phases) is directly proportional to its affinity/dynamic equilibrium with the stationary phase
• Application of bio-mimetic chromatography reduces animal testing and late-stage attrition, lowers candidate selection cost, and allows early dose estimation
• We have modified the original methodology by Dr. K. Valko to implement it for high-accuracy lung tissue binding detection [1]. It has been successfully validated, using experimental literature data for known compounds.

References

1. Valko K. Physicochemical and biomimetic properties in drug discovery: chromatographic techniques for lead optimization. – John Wiley & Sons, 2013.
2. Valkó, K., Nunhuck, S., Hill, A.P. (2011) Estimating unbound volume of distribution and tissue binding by in vitro HPLC based human serum albumin and immobilized artificial membrane-binding measurements. Journal of Pharmaceutical Sciences, 100, 849–862.