Presentation by Sirius Analytical covering measurement of pKa, LogP, LogD, Solubility, Supersaturation and precipitation kinetics. For more details visit www.sirius-analytical.com

1. Sirius Analytical Measuring pK a s, logP and Solubility by Automated titration Jon Mole Technical Sales Manager www.sirius-analytical.com

6. pK a is the pH at which an ionisable group is “half-ionised” / 70 Propranolol (a base): pK a = 9.53 BH + B BH + B Flumequine (an acid): pK a = 6.27 HA A - HA A -

8. / 70 2 or more acidic groups, no basic ~ 3% 1 basic group, no acidic ~ 42% 1 acidic group, no basic ~ 12% Others ~ 3% 1 basic group + 2 or more acidic ~ 3% 1 acidic group + 2 or more basic ~ 4% 1 acidic group + 1 basic ~ 8% 2 or more basic groups, no acidic ~ 25% With thanks to Tim Mitchell and Ryszard Koblecki, Millennium Pharmaceuticals Ltd. 32,437 Ionizable drugs in World Drug Index (63% of total)

9. Human Gastrointestinal (GI) Tract / 70 STOMACH 0.1 m 2 DUODENUM 0.1 m 2 JEJUNUM 60 m 2 ILEUM 60 m 2 COLON 0.3 m 2 pH (fasted) 4.6 (2.4 - 6.8) 6.1 (5.8 - 6.2) 1.7 (1.4 -2.1) 6.5 (6.0 - 7.0) 6.5 8.0 5.0 - 8.0 pH (fed) 5.0 (0.1 hr) 4.5 - 5.5 (1 hr) 4.7 (2 hr) 6.5 8.0 3-4 h small Intestine transit time

10. LogP and logD describe lipophilicity / 70 P = partition coefficient. The ratio of concentrations of unionised species dissolved in two immiscible solvents (e.g. water + octanol) which are in equilibrium. D = Distribution Coefficient. The ratio of all species dissolved in two immiscible solvents which are in equilibrium. P is constant D is pH-dependent

11. Lipophilicity profiles are pK a and pH dependent / 70 Desipramine pK a = 10.14 Diphenhydramine pK a = 8.26 Triamterene pK a = 3.92 These molecules all have similar value for log D at pH 7.4. Their lipophilicity profiles are quite different Flat part of curve: log D = log P of neutral species Diclofenac pK a = 3.99 Phenobarbital pK a = 7.43 Nifuroxime pK a = 10.56 Big changes in lipophilicity occur over physiological pH range Physiological pH range

12. / 70 Why is logP (and logD) important? LogP and logD (lipophilicity) provide a rough guide to pharmacokinetic behavior. LogD at pH 7.4 Implications for drug development Below 0 Intestinal and CNS permeability problems. Susceptible to renal clearance. 0 to 1 May show a good balance between permeability and solubility. At lower values, CNS permeability may suffer 1 to 3 Optimum range for CNS and non-CNS orally active drugs. Low metabolic liabilities, generally good CNS penetration 3 to 5 Solubility tends to become lower. Metabolic liabilities increase Above 5 Low solubility and poor oral bioavailability. Erratic absorption. High metabolic liability, although potency may still be high.

19. Titration cell for SiriusT3 Capillaries, for adding reagents pH electrode, diameter 3mm Glass vial, 4 ml total capacity Electronic thermometer Automatic overhead Stirrer Probes require a minimum of 0.5mL of solution contained in glass vial. Typical assay volume = 1ml. UV Dip Probe. / 70

21. A refined solution / 70

25. 3-D spectrum: pH vs. absorbance vs. wavelength pK a from UV spectra / 70

26. pK a from UV spectra / 70

27. pK a result calculated by Target Factor Analysis (TFA) / 70 pKa Result

31. Detecting precipitation SiriusT3 has built in turbidity detection . Shaded area shows pH where sample precipitated. This is a warning, do not use this data to determine pK a of miconazole! Repeat in cosolvent to avoid precipitation and get reliable pK a data. / 70

33. Principles of pH-metric logP measurement A solution of the sample is titrated in a two-phase system (water + octanol) The sample can ionise in water (pK a ), or it can partition into octanol (logP) The presence of the octanol disturbs the pK a equilibrium. The pK a shifts to a new value (p o K a ) to minimise this disturbance. We calculate the logP from this shift in pK a . / 70

34. Flumequine (acid) pK a = 6.27, p o K a = 7.99 log P = 1.72 Titrations with equal volumes of water and octanol / 70 Lipophilicity profiles: these profiles are correct for high logD, but do not show partitioning of ionic species Diacetylmorphine (base) pK a = 7.95, p o K a = 6.37 logP = 1.58 Aqueous pK a p o K a

37. Validation of UV pK a method R.I. Allen, K.J. Box, J.E.A. Comer, C. Peake, K.Y. Tam, J. Pharm. Biomed. Anal., 17 , 699-712, 1998. K.Y. Tam, K. Takács-Novák, Pharm. Research,. 1999, 16, 374-381 R.C. Mitchell, C.J. Salter, K.Y. Tam, J. Pharm. Biomed. Anal., 1999, 20, 289-295 K.Y. Tam, M. Hadley, W. Patterson, Talanta, 1999, 49, 539-546 pK a (spec) = 1.006 x pK a (pH-metric) n = 31 R 2 = 0.999 RMSD = 0.098 Benzoic acid (3.98) Icotidine (3.29, 5.39, 6.22, 9.97) Lupitidine (2.79, 5.96, 8.25, 9.66) Nicotinic acid (2.10, 4.63) Nitrazepam (2.90, 10.39) Niflumic acid (2.28, 4.86) m-aminobenzoic acid (3.17, 4.54) p-aminosalicylic acid (1.79, 3.58) Phthalic acid (2.70, 4.86) Phenol (9.73) Phenolphthalein (8.87, 9.35) Pyridoxine (4.90, 8.91) Quinine (4.33, 8.59) SB-221789 (2.74) SKF-75250 (1.48, 6.59) (measured at 25ºC and an ionic strength of 0.15 M) / 70

38. Validation of pH-metric logP method B. Slater, A. McCormack, A. Avdeef and J.E.A. Comer, J. Pharm. Sci . 1994, 83 ,1280-1283 J.E.A. Comer, K. Chamberlain and A. Evans in J. Devillers (Ed.), SAR QSAR Environ. Res., Vol.3 Issue 4; Molecular Descriptors , Gordon and Breach, Philadelphia 1995, pp. 307-313. K. Takács-Novák and A. Avdeef, J. Pharm. Biomed. Anal. 1996, 14 ,1405-141; 61 samples over eight logP units amino acids, peptides, ampholytes, barbiturates, ß-blockers, herbicides, phenols, various others Graph plotted using Polyfit program from RefinementPro 2 / 70

42. Starting the CheqSol Assay - Seeking precipitation Precipitation causes light scattering, and system detects this as an increase in the light absorbed. Kinetic solubility determined at point of precipitation. Before precipitation, no light is absorbed by the solution Absorbance 0.0 0.8 1.6 2.4 3.2 200 300 400 500 600 700 / 70 Solid added to vial. (5 to 20mg on GLpKa) (0.5 to 2mg on SiriusT3) Instrument adds water (or water-cosolvent), then adjusts pH to dissolve sample. Solution titrated towards the pH where the sample becomes neutral. Eventually it precipitates After precipitation, most light is scattered Wavelength (nm) 0.0 0.8 1.6 2.4 3.2 200 300 400 500 600 700

44. Understanding the Bjerrum Graph / 70 Sample = base with one pK a Precipitate is present pH 0.0 0.5 1.0 2 4 6 8 10 12 pH = pK a Sample is unionised at this pH Sample is ionised at this pH Bj =Moles of bound H + ions per mole of sample Precipitation Bjerrum Graph. For a base with one pK a , Solution Bjerrum Graph. For a base with one pK a , 1.0 B BH + pH 2 4 6 8 10 12 % Species 0 50 100

59. Can we predict whether a sample is a non-chaser? Imipramine Non-chaser Amitryptyline Non-chaser Chlorpromazine Non-chaser Desipramine Non-chaser Nortriptyline Non-chaser Maprotiline Chaser Secondary and tertiary amines with logP > 4. Chlorprothixene Non-chaser converts to chaser Similar structures, but maprotiline contains a -CH 2 -CH 2 - bridge. Non-chaser Trimipramine / 70

60. More non-chasers.….. Amiodarone Verapamil Quinine Diltiazem Deprenyl / 70

61. ..….. and some chasers Terfenadine Nadolol Loperamide Metoclopramide Amodiaquin Pyrimethamine / 70

62. Investigating precipitation and dissolution behaviour Piroxicam Sulfamerazine Supersaturated acidic sample Subsaturated acidic sample Subsaturated acidic sample Supersaturated acidic sample Most of the early compounds we investigated show a tight symmetry. / 70

63. Investigating precipitation and dissolution behaviour Papaverine Furosemide Supersaturated acidic sample Subsaturated acidic sample Subsaturated basic sample Supersaturated basic sample Some compounds show a clear offset! / 70

64. Using the Precipitation Rate graph to investigate ~100 ionisable drugs, we have found that there appears to be four classes of behaviour. The Four Class Model / 70 Slow Precipitator Fast Precipitator Slow Dissolver “ Chasers” Slow rate for both precipitation and dissolution Examples: Ibuprofen, Benzocaine, Benzthiazide. “ Non-Chasers” Fast rate of precipitation, slow rate of dissolving Examples: Nortriptyline, Amitryptyline, Imipramine. Fast Dissolver “ Super Dissolvers” Slow rate of precipitation, Fast rate of dissolving Examples: Tolmetin, Papaverine, Chlorzoxazone. “ Ghosts” Fast rate for precipitation and dissolution Examples: None yet discovered.

65. CheqSol Technology A weak base might dissolve fully in the stomach but precipitate on entering the high pH environment of the upper intestinal tract. Can the patterns we observe in CheqSol be used to identify which formulation/delivery methods can be used to improve bioavailability? Does the supersaturation exhibited by “chasers” mean that the bioavailability is already enhanced over what the thermodynamic properties imply, and thus further formulation/delivery work is unwarranted? Do non-chasers fall out of solution as amorphous material whereas chasers produce crystalline precipitate? Amorphous materials are amenable to solid state dispersion nanoparticle delivery methods. Alternatively, could a formulation technique be used to keep a supersaturated sample in a supersaturated state for longer than expected? Are the properties we observe inherent to the compound, or can they be changed by the use of excipients, milling techniques etc.? CheqSol is a unique tool for investigating the precipitation characteristics of a drug. / 70

66. Validation of CheqSol solubility method CheqSol vs. Shake-flask results for compounds 1– 14 Six replicate shake-flask experiments compared with six CheqSol experiments for each compound Box, K J. Völgyi, G. Baka, E. Stuart, M. Takács-Novák, K. Comer, J E A. J. Pharm. Sci. 2006, in press / 70 19 compounds in this group – see next slide