Compound optimization after HTS: more than just potency 

(Kurt R. Brunden)

Definitions and concepts:

  • Probe compound vs. drug candidate – A research probe compound is developed for proof-of-concept (POC) studies in animal disease models or as a potential candidate for an Investigational New Drug (IND) application. Requires less characterization than a true drug candidate
  • hERG potassium channel – involved in cardiac electrical activity, results in a fatal disorder (long QT syndrome) when inhibited; needs to be avoided as a side effect during drug discovery
  • Cytochrome P 450 (CYP450) – large group of catalytic enzymes involved in drug metabolism, accounting for ~80% of total small molecule drug metabolism; most drugs are metabolized by 6 isozymes out of the 50+ total
  • Contract research organization (CRO) – organizations for outsourced drug discovery services; range from large full-service providers to niche specialty groups
  • Good Laboratory Practices (GLP) – regulatory principles by which research studies are planned, performed, monitored, recorded, reported, and archived; aims to improve the quality and reliability of data
  • Liver microsomes – preparation of ER vesicles obtained after low speed centrifugation of homogenized liver preps; contain major metabolic enzymes including CYP450 enzymes
  • Investigational New Drug (IND): Approval by the FDA to take a drug into human clinical testing

Probe compounds – for POC research in animal models, requires:

  • Reasonable ADME properties (solubility, clearance, half-life, brain penetration)
  • Demonstration of adequate free drug levels in the brain
  • Safety in animal species of choice (mouse/rat) at projected efficacy doses
  • Evidence that compound metabolism doesn’t change upon repeated dosing

An IND candidate requires a more rigorous analysis, including all of the above plus:

  • In vivo safety pharmacology, including hERG and human CYP450 inhibition profiling
  • human microsome studies to determine predicted clearance, CYP metabolism and preliminary metabolite identification
  • IND-enabling studies (via CROs), including GLP; respiratory, cardiovascular, and safety toxicology in two species

With relatively little expense, academic labs can perform preliminary safety pharmacological assessments that will provide important information about whether a compound (or compound series) has drug candidate potential.

  • An investment in these studies early on could lessen attrition at later stages.

Compound solubility is affected by many factors including pH, salt forms, buffer systems, etc.

  • Sufficient solubility is necessary to:
    • Generate interpretable results in assay buffer systems
    • Allow compound administration in animal studies (by any administration route)
  • General rule of thumb: > 60 ug/ml or > 100 uM solubility
  • Two basic types of solubility determinations:
    • DMSO stock dilutions – measures compound precipitation (typically a kinetic measurement). Many drug discovery labs determine solubility by diluting from a stock DMSO solution into aqueous systems; measurements are taken with a UV/Vis spectrometer (light scatter of precipitates).
    • solid compound – measures compound dissolution (typically an equilibrium measurement)

LC-MS/MS (liquid chromatography–mass spectrometry) is a highly recommended investment purchase for academic drug discovery labs.

  • Key ADME properties can be analyzed with LC-MS/MS:
    • Plasma PK (e.g. clearance and half-life determinations)
    • Brain penetration
    • Approximation of free drug levels in plasma and brain (compound non-specific binding to plasma/brain proteins)
    • Approximation of human metabolism using liver microsomes (including identification of major CYP450 isozymes involved in the compound’s metabolism)

BBB penetration

  • A brain/plasma ratio ≥ 1 indicates full BBB penetration.
  • Compounds with <1 ratios might still equilibrate across the BBB (free non-protein bound compound). A free compound may be fully BBB permeable but still have a ratio < 1 if plasma non-specific binding is greater than in the brain.
  • The unbound fraction in plasma and brain can be approximated by equilibrium dialysis.
    • Most brain-penetrant compounds show free B/P ratios of 0.9-1.1.

Predicted human metabolism

  • Metabolic rate can be estimated by mixing the compound with human liver microsomes, which contain CYP450, flavine monooxygenase (FMO), and UDP-glucuronosyl transferase (UGT) metabolic enzymes. Compound metabolites are analyzed at various time points with LC-MS/MS.
  • The slope of compound decay can be used to determine the intrinsic clearance, which can be compared to the human hepatic blood flow to get a sense of relative compound stability.
  • The CYP450 isozymes involved in the compound’s metabolism can also be identified from microsome studies by analyzing decreases in metabolism upon addition of specific CYP inhibitors.
    • See FIG. 1 for common CYP450 isozymes involved in drug metabolism.
  • Significant metabolism by CYP2D6, a member of the CYP450 family, is a red flag due to allelic variation (see example in Part I).

Significant inhibition of major CYP450 enzymes can result in undesirable drug-drug interactions.

  • If the objective is to identify an IND candidate compound, a large number of safety and toxicology studies are needed. Certain safety analyses are practical and inexpensive enough for an academic group to conduct and will help determine if a compound is worth pursuing as a drug candidate.
  • CYP450 inhibition profiling can be simply assayed by:
    • LC-MS/MS (measure inhibition of known CYP450 substrates using baculozomes)
    • Commercial kits (e.g. Invitrogen Vivid fluorescent kits)
  • The extent to which CYP450 inhibition will prove problematic depends on the strength of interaction the compound with the CYP, and the relative concentration of the compound in the blood at efficacy doses.
    • Compounds with >75% CYP450 inhibition at 10uM should be flagged.
  • FDA guidance suggests that in vivo CYP450 inhibition studies are needed if plasma Cmax is >10% of CYP450 Ki.

hERG binding assays are another type of safety analysis that most labs can perform.

  • At least 11 drugs have been withdrawn from the U.S. as a result of their likely hERG cardiac channel inhibition.
  • Commercial ligand binding kits are available (e.g. Invitrogen Predictor hERG FP assay kit).
    • hERG binding assay generally have good correlation with the more definitive patch clamp analyses.
  • Goal of ~100-fold window between effective plasma drug concentrations and hERG Ki/IC50.
    • Compounds with >50% inhibition at 10 μM should be flagged.
  • As with CYP450 inhibition, the extent to which hERG interaction is problematic will depend on the required drug concentration in blood and the binding constant to hERG.

Rodent tolerability studies

  • Most academic research institutions are equipped to conduct rodent efficacy studies and non-GLP preliminary rodent toxicological assessments.
  • Before a compound progresses into efficacy testing in animal models of disease, it is important to establish that the compound can be safely tolerated by the animal.
  • Relatively simple testing can determine maximum tolerated dose (MTD) and repeated dose tolerability, prior to efficacy testing.
    • MTD design: Normal mice (n=4) are dosed via oral gavage (0.5% methylcellulose) starting at 3 mg/kg, with 3X dose-escalation every two days until two or more mice show signs of intolerance (altered locomotor activity, sedation, ataxia, hypo- or hypertonia, salivation or excitation).
    • 1 month tolerability design: Normal mice (n=4/dose) are dosed at 0.1x-, 0.3x- and 1x-MTD via drinking water for 2 weeks or 1 month (assumes 5 ml/mouse/day). Assessments include: behavioral observations, body weights, organ weights at study completion, complete blood counts at study completion, plasma and brain compound levels at study completion (compared to separate group receiving drug for 3 days).
  • For IND-supporting studies, academic labs should seek CRO assistance.
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