A fitting Drug Metabolism and Pharmacokinetic (DMPK) profile remains a challenge to reduce risks and improve efficiency in pharmaceutical R&D, leading to major drug failure cases. Compounds with optimal DMPK results seem to be efficacious and safe when choosing the same for drug development.

For the last 32 years, pharmaceutical industries have witnessed the vital role of DMPK and the science behind it, supporting and facilitating drug discovery and development process. Its predominant contribution is identifying the best drug candidates in Lead Identification and Optimization phases to enter Development phase. DMPK studies in preclinical phase can be broadly classified into in vitro DMPK and in vivo DMPK studies.

Nowadays, Electronic Laboratory Notebooks (popularly known as an ELNs) take huge credits for supporting DMPK studies. No wonder that this software system, which has been specially fabricated for scientific studies, has played a tremendous role in accelerating the preclinical studies by capturing, calculating, organizing, interacting, storing, searching and reporting scientific data across the scientific spectrum. Being a cloud-based software, it helps scientists to go paper-free.

In in vitro DMPK study there are a few sets of highly qualified assays which ensure the safety and efficacies of the drugs tested. These assays are configured as workflows in ELNs from High-throughput Screening to Lead Optimization phases. ELN assists the in vitro DMPK profile by providing a broad platform for the interaction and integrations of number of databases, instruments and other scientific setups which facilitate the process.

Mentioned below are the assays and their purposes along with an ELN’s role in each of them:

Assay Purpose of the Assay Instruments Role of ELN
Physicochemical screening Assays
  • Aids in the reduction of lipophilicity
  • Modifies solubility
  • Screens the best suited physicochemical properties of the desirable drug
  • LC-MS/MS
  • Performs all physicochemical calculations
  • Batch file generation feature – sends data to plate readers or sophisticated instruments (e.g. LC-MS)
  • Imports LC-MS exports (Excel or CSV files) for final solubility assessment of drugs
Drug-transporter interaction Assays
  • Helps to understand the compound’s absorption potential by assessing membrane permeability with transformed human intestinal enterocytes (CaCo2 cells) / Madin-Darby canine kidney (MDCK) cells / Parallel artificial membrane permeability assay (PAMPA)
  • Nanoelectrospray MS/MS
  • Calculates Permeability coefficient (Papp) and Efflux ratio
  • Knocks out unexpected values for further calculations
Transporter screening Assays
  • Activation or inhibition of a drug transporter protein that involves in the ADME and toxicity of a drug (could have considerable clinical effects ranging from lowering drug efficacy to compromising safety)
  • Fluorescence and Radioactivity probes
  • Fluorescence spectrometers coupled with LC/MS
  • Determines IC50 values
  • Generates reports by consolidating experiments (created in ELN)
  • Allows consolidation of results for future reference or investigational new drug application
Metabolic Stability Assays
  • Metabolic Stability assays establish the percentage of compound’s metabolism over time using microsomes or hepatocytes
  • Helps to compare and assess the compound’s metabolic rate and metabolites generated across species to fetch the PK values
  • LC-MS/MS is regarded as the best biological analytical method for the Metabolic Stability assays
  • Calculates intrinsic clearance of tested drugs
  • Senior scientist can review the experiments done, if necessary
  • Helps in considering only reviewed and approved experiments for further studies
Metabolite identification Assays
  • Metabolite Identification assays optimize the chemical structure based on propensity to metabolic activation.
  • This may increase or decrease the chances of unfavorable protein interactions
  • High Resolution Mass Spectrometry (HRMS)
  • View chemical structure of any registered drug in the database
  • Create chemical structures and perform chemical reactions in order to identify the desired and unwanted metabolites
In-vitro CYP450 inhibition and induction screens Assays
  • Used to assess the potential of the compound towards drug-drug interactions
  • Cytochrome P450 induction assay recognizes whether the test compounds have the potential to induce CYP1A2, CYP2B6 or CYP3A4 in cultured human hepatocytes by assessing mRNA levels
  • P450 Inhibition assay, a reduction in the generation of the metabolites is examined
  • LC-MS for quantification of CYP proteins
  • qRT-PCR is used for capturing relative mRNA expression levels for CYP1A2, CYP2B6 and CYP3A4
  • Easily compares assay’s fold induction data that is normalized to vehicle control: positive control responses
  • Calculates IC50, EC50 and Emax
Plasma Protein Binding (PPB) and Blood Partitioning Assays
  • Helps to understand how binding to plasma impacts the distribution of a drug into various tissues in the body
  • Used to evaluate the blood to plasma ratio which determines the concentration of the drug in whole blood compared to plasma and gives an indication of drug binding capacity to erythrocytes
  • Equilibrium dialysis combined with LC-MS for measurement of PPB in drug discovery
  • Lab on chip (LOC) is recommended to carry micro fluids management
  • Determines a value of the fraction of compound unbound to proteins (another essential parameter to be explored in-vitro)
  • Calculates blood to plasma ratio
  • Generates and prints labels to deal with many samples and differentiate the same
The hepatotoxicity and cytotoxicity Assays
  • Measures cell death due to the cytotoxic effect of a test compound
  • Cytotoxicity is assessed using MTT technique (calorimetric assay)
  • Heat map configuration assists interpretation of these assays based on varied color range

The results obtained from the assays for the list of tested compounds along with study parameters can easily be sent to various biological and chemical databases and stored using ELN. Also, data can easily be searched and mined for further studies to proceed with in vivo DMPK studies.

Emergence of new wet lab methods, techniques and lab automation technologies have impacted DMPK tremendously. Some of them are 3D cell cultures, CRISPR-Cas9 system, RNA interference (RNAi) screens, Hamilton automated robotics, microfluidics, screening using Artificial Intelligence and Machine Learning. Apart from LC-MS, Nanoflow liquid chromatography mass spectrometry (Nc–MS), High Resolution Mass Spectrometry (HRMS) and lab-on-a-chip (LOC) instruments perform a vital role in in vitro DMPK studies.

To meet the advancement in lab technologies, ELN platforms and solutions are constantly updated and upgraded to cater to all aspects of DMPK studies in present and future.

Stay tuned to know the role of ELN in In vivo DMPK studies.

In vitro DMPK Assay

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