4) Procedure

The following steps outline the detailed procedure for ADME screening in early drug discovery:

1. Step 1: Selection of Compounds for ADME Screening
   1. Select compounds based on their potential to interact with a biological target and their chemical diversity. Choose compounds from high-throughput screening hits or lead candidates that are structurally diverse and show promise in initial biological assays.
   2. Ensure that selected compounds represent a wide range of chemical properties to provide a thorough understanding of their ADME characteristics.
   3. Ensure that the selected compounds are chemically stable and suitable for testing in ADME assays, with known purity and structural integrity.
2. Step 2: Absorption Screening
   1. Conduct in vitro absorption studies to evaluate the compound’s permeability across biological membranes. This can be done using models like Caco-2 cell lines, PAMPA (Parallel Artificial Membrane Permeability Assay), or rat intestinal perfusion.
   2. Measure the compound’s permeability, solubility, and transport across the intestinal membrane. Focus on key metrics such as the Papp (apparent permeability coefficient) value to assess the compound’s absorption potential.
   3. Assess the compound’s potential to be absorbed via oral administration, based on its permeability, solubility, and active transport properties.
3. Step 3: Distribution Screening
   1. Perform protein binding studies to assess the extent to which the compound binds to plasma proteins, which can affect its distribution in the body. This can be done using techniques such as ultracentrifugation or equilibrium dialysis.
   2. Use in vitro models to evaluate tissue distribution and the compound’s ability to cross biological barriers such as the blood-brain barrier (BBB), if relevant to the therapeutic target.
   3. Evaluate the compound’s distribution profile using animal models or computational methods to predict how it may distribute in different tissues, organs, or compartments of the body.
4. Step 4: Metabolism Screening
   1. Assess the metabolism of the compound using human liver microsomes, hepatocytes, or recombinant enzymes to determine its metabolic stability and the enzymes involved in its biotransformation (e.g., cytochrome P450 enzymes).
   2. Determine the compound’s half-life (t1/2) in hepatic microsomes or hepatocytes to predict its metabolic stability.
   3. Perform studies to evaluate potential drug-drug interactions based on the compound’s interaction with key enzymes (e.g., cytochrome P450) that are involved in the metabolism of other drugs.
5. Step 5: Excretion Screening
   1. Assess the compound’s excretion profile by determining the route of elimination, either through urine, feces, or bile. This can be evaluated using radiolabeled compounds or mass spectrometry to track the compound in animal models.
   2. Evaluate the compound’s renal clearance and potential nephrotoxicity, if relevant. This can be performed using in vitro assays or animal models to assess the compound’s potential to cause kidney damage.
   3. Monitor the compound’s pharmacokinetics (PK) in vivo, including its half-life, clearance rate, and bioavailability, to better understand its absorption and elimination characteristics.
6. Step 6: Data Analysis and Interpretation
   1. Analyze the ADME data to evaluate the pharmacokinetic properties of the compound. Identify any potential issues such as poor bioavailability, excessive plasma protein binding, rapid metabolism, or toxicity risks.
   2. Use the data to inform decisions about compound optimization. Modify the chemical structure of the compound to improve its ADME properties, such as increasing solubility, reducing clearance, or enhancing tissue distribution.
   3. Collaborate with medicinal chemists to apply SAR (Structure-Activity Relationship) to optimize the ADME profile based on experimental results.
7. Step 7: Documentation and Reporting
   1. Document all ADME screening results, including data from absorption, distribution, metabolism, and excretion studies. Ensure all raw data, analysis, and interpretations are recorded accurately.
   2. Prepare an ADME Screening Report that includes detailed information on the methods used, results obtained, and conclusions drawn regarding the drug-likeness of the compound.
   3. Ensure that all data is stored securely and can be accessed for regulatory compliance and future use in drug development.

5) Abbreviations

• ADME: Absorption, Distribution, Metabolism, Excretion
• PK: Pharmacokinetics
• Papp: Apparent Permeability Coefficient
• IC50: Half-Maximal Inhibitory Concentration
• LD50: Lethal Dose for 50% of the population

6) Documents

The following documents should be maintained throughout the ADME screening process:

1. ADME Screening Report
2. Absorption and Permeability Data
3. Protein Binding and Distribution Data
4. Metabolism and Excretion Data
5. Compound Modification and Optimization Logs

7) Reference

References to regulatory guidelines and scientific literature that support this SOP:

• FDA Guidance for Industry on Drug Discovery
• Scientific literature on ADME testing methodologies and in vitro assays

8) SOP Version

Version 1.0: Initial version of the SOP.