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Physicochemical Properties at Bayer HealthCare (Wuppertal) and Their Use in Medicinal Chemistry

Physicochemical Properties at Bayer HealthCare (Wuppertal) and Their Use in Medicinal Chemistry . Contents. Measured physico-chemical parameters Introduction of our laboratory Model systems for lipophilicity Solubility Use in medicinal chemistry. Physicochemical Properties Measured.

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Physicochemical Properties at Bayer HealthCare (Wuppertal) and Their Use in Medicinal Chemistry

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  1. Physicochemical Properties at Bayer HealthCare (Wuppertal) and Their Use in Medicinal Chemistry

  2. Contents • Measured physico-chemical parameters • Introduction of our laboratory • Model systems for lipophilicity • Solubility • Use in medicinal chemistry

  3. Physicochemical Properties Measured Lipophilicity Membrane Affinity MA Plasma binding human serum albumin binding HSA rat serum albumin binding RSA pKa pKa Solubility screening in various buffers SOL equilibrium in buffer equilibrium in galenic formulations

  4. Logistics for HT Physicochemistry BLJ input for sample identification Vial collecting rack Bar-coded vial for sample registration

  5. cMA, cHSA Solubility HSA-binding Membrane affinity Data Handling by Laboratory Information and Management System (LIMS) Chemistry Laboratories BAYNO/Prepno., Barcode, Scale of Tests, Weight, Molecular Weight, Principle investigator, Projekt, Comparison Lab.- PDH PIX Journal flag for bases or acids Sample Sample- registration Reports PILO-LIMS Results, Calibration Data Rackno. Rack Position Area, Barcode Time Methods Sequences Archive LC/MS/MS Roboter LISSY Sample- Waters Quattro-Micro Bar-code scan preparation Rack MTP

  6. Our model system Solid-supported lipid membranes (TRANSIL)

  7. mlogP vs mlogMA 6.0 5.0 4.0 logP 3.0 2.0 1.0 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 mlogP (Österberg) mlogP (Österberg) acid mlogD7.4 vs mlogMA mlogP (Österberg) base 4.0 3.0 2.0 1.0 0.0 -4.0 -2.0 0.0 2.0 4.0 6.0 mlogD -1.0 -2.0 -3.0 -4.0 -5.0 -6.0 logMA Why Use Membrane Affinity? 1. comparison with other lipophilicity descriptors

  8. Why use membrane affinity? 2. comparison with physiological membranes Influence of cholesterol content has to be considered

  9. Why use membrane affinity? Influence of cholesterol C. Tradum et al.:Biophysical J. 78 2496-2492

  10. Why use membrane affinity? Influence of cholesterol

  11. Why use membrane affinity? Influence of cholesterol • Influence on passive permeation expected to be strong • flexibility of plasma membrane is strongly influence by cholesterol, content usually about 80 mol% of phospholipids content • Influence on distribution expected to be low • 80% of all membranes are intracellular with a cholesterol content about 4 mol% of phospholipids content

  12. Comparison of solubility methods • precipitation • all compounds • small amounts • fast analytics • compound dissolved in organic solvent • oversaturated solutions possible • from powder • selected compounds • large amounts (two samples) • specific analytics • sensitive to morphology • sensitive to purity • sensitive to solvent impurities

  13. Comparison of solubility methods • precipitation • Dissolve compound in DMSO (2mg/40µl) • Add 10µl of this solution to 1000µl buffer (1% DMSO) • Shake for 24h at room temperature • Centrifuge to get supernatant • Establish LC/MS/MS method • Measure calibration standards and probe • from powder • Weight an appropriate amount of compound as solid • Add 1000µl buffer • Shake for 24h at room temperature • Centrifuge to get supernatant • Establish LC/MS/MS method • Measure calibration standards and probe

  14. Comparison of solubility methods EXAMPLES:

  15. O H O O O O H N N O H H Cl N O N S H N O O 2 O + N O O O O O N H O O O H Cl O H N H N N H O O S O O O H O O N O Cl H H N N S H N N N N H O N O O Cl F N H O O N H N H O H O O O N H Comparison of solubility methods charged neutral Yalkowsky Name MOLSTRUCTURE compounds (water) 40 Warfarin 235 500 Primidone 60 Metolazone 88 6 Nifedipine 9 140 Ketoprofen 275 4 Glyburide 1.1 8.6 Indomethacin 240 11000 Cimetidine 800 Phenacetin 1080 14 Haloperidol 180 26 Phenytoin 23 36 Ibuprofen 290 6300 Acetanilide

  16. Lessons learned from solubility comparisons • method differences not really critical • physical form very important differences between research and development result from: • morphology differences • impurities • solvent content • counter-ions and buffers are important when compound is charged in solution

  17. Case Histories: The Use of Physicochemical Properties Two different projects as examples: 1. Reducing lipophilicity and HSA binding to increase fraction unbound: erectile disfunction 2. Influence of solubility on in vivo efficacy: the HSV project

  18. Reducing Lipophilicity and Protein Binding to Increase Fraction Unbound Starting point: initial compound moderate effective IC50 PDE-5: 530nM DP1 compound: Vardenafil highly effective IC50 PDE-5: 2nM Insufficient physicochemical properties: high membrane affinity: 16500 high protein binding: 1.7e-5 mol/l solubility: below detection limit fraction unbound: <1% no in vivo efficacy improved physicochemical properties: reduced membrane affinity: 580 reduced protein binding: 1.2e-4 mol/l solubility: 220 mg/l fraction unbound: 14% excellent in vivo efficacy

  19. Reducing lipophilicity and protein binding to increase fraction unbound Even the prediction of the in vivo effect from IC50 and fraction unbound (calculated from MA and HSA) was possible 1.E+02 initial compound 1.E+01 Dose measured in vivo 1.E+00 1.E-01 Vardenafil 1.E-02 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 Dose calculated from IC and physicochemical 50 properties

  20. 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.00 0.01 0.10 1.00 10.00 100.00 Influence of solubility on in vivo efficacy Starting point: Example 1 moderate activity IC50: 750nM survival % of HSV infected mice at 60mg/kg vs free serum normalized with IC50 survival % at 60mg/kg Physicochemical properties: Membrane affinity: 1430 protein binding: 2e-4 mol/l fraction unbound: 10% Solubility: 17mg/l optimization of physicochemistry not necessary, activity has to be improved free serum/IC50

  21. survival % of HSV infected miceat 60mg/kg vs free serum normalized with IC50 1.0 0.9 0.8 0.7 0.6 survival % at 60mg/kg 0.5 0.4 0.3 0.2 0.1 0.0 0.00 0.01 0.10 1.00 10.00 100.00 free serum/IC50 Influence of solubility on in vivo efficacy Example 2: brilliant compound in vitro IC50: <1 nM Development candidate in vitro activity IC50: 20 nM Physicochemical properties: Solubility: <0.1 mg/l excellent in vivo efficacy when administered as solution, no in vivo efficacy even as micronized powder Physicochemical properties: Membrane affinity: 1590 protein binding: 1e-5 mol/l fraction unbound: 1% Solubility: 2.7mg/l good in vivo efficacy

  22. Conclusion • Impact of Physicochemistry Proven • Physicochemistry/ADME Implemented in Medicinal Chemistry • Properties Routinely Measured for Every Strategic Project • Use in Lead Optimization and Exploratory Research Established

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