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Developing Best Practice Strategies to Generate 

Reliable Solubility Data and Make Efficient Informed 

Decisions on the Most Appropriate Solid Form. 

Brian Frøstrup, Head of Preformulation, NeuroSearch

Developing Best Practice Stategies to Generate Reliable Solubility Data 

and Make Efficient Informed Decisions on the Most Appropriate Solid Form 

Agenda 

– About NeuroSearch 

– Thermodynamic background of solubility 

– Determination of solubility. 

– Shakeflask 

– Nephlometric (kinetic) 

– Potentiometric 

– Solubility of salts 

– Examples of in-vivo exposure and solubility data 

– Conclusion 

6 th Annual Improving Solubility for Pharmaceutical and Biologics. 21 st -23 rd June 2010. 2

Who is NeuroSearch ? 

Key competence: CNS diseases 

– Ion channel agonists, antagonists and modifiers 

Small molecules 

Clinical development pipeline: 8 programmes 

– Huntexil Phase III orphan drug for huntingtons disease. 

– Tesofensine for Obesity about to enter Phase III 

Big pharma partners: Eli Lilly, Johnson & Johnson and 

Abbott 

Cash position: >120 Mill. Euro 

Organisation: ~220 employees in DK and S 

Head-quater in Ballerup 8 miles from downtown CPH 


NeuroSearch Pipeline 


Solubility. Thermodynamic background 

Solubility is defined by Gibbs free energy, G 

G a, Solution increases as activity (concentration) increases 

0 

G G RT ln( C) 

Where is the activity coefficient 

a, Solution a, 

Solution 

 

Dissolution process: 

a a( 

solution ) 

s 

G 

At equlibrium at constant T. 

G 

 

G 

a( s) 

a( 

solution ) 

Ga, 

solution 

Ga, 

solid 

G 

 

a( 

s) 

a( 

solution ) 

a, 

solution 

G 

a, 

solid 

Higher Gibbs free energy for 

solid e.g amorphous -> 

Solubility increases 

 

0 

G 

0 

a, 

solution 

0 

RT ln( C 

saturated 

) 

a(sol) 

a(s) 


Solubility. Shake flask method 

Classical method 

Also known as thermodynamic solubility 

Solubility at equilibrium ~ T ∞ 

Pros: 

True solubility 

Well defined solid phase 

Accurate, HPLC-UV 

Impurity/degradation profile 

Versatile and adaptable 

Covers the whole pH range 

Organic solvents 

Dosing vehicles 

Cons: 

Labour intensive 

Low-throughput ~ 10-50 cps per week 

Time consuming 

Equlibration 

HPLC mthd dev. Semi standardised 

External standard curve necessary 

Solubility of cmp in e.g MeCN 

Cmp demanding 10-100 mg 

LOD ~ 0.5 µg/ml (HPLC-UV) 

6 th Annual Improving Solubility for Pharmaceutical and Biologics. 21 st -23 rd June 2010. 6 17 June 2010

Solubility. How to perform a Shake flask experiment 

pH 

Measure 

Separate 

supernatant 

Equilibrate 

e.g.1 ml vial for 

72 h 

Solid phase 

XRPD, DSC 

Dilute 

Report 

-Solubility 

-pH 

-Solid phase 

HPLC 

Quantification 


Solubility. Nephlometric solubility 

High throughput 

Also known as kinetic solubility 

Precipitation from solution 

Pros: 

High throughput, automated 

Low manning costs 

Relevant for in-vitro screening 

Keeping cmp in solution 

No need for external calibration 

Low cmp spendature 1-10 mg 

Stock solutions are used 

E.g 30 mM DMSO stock 

already made for in-vitro 

Lipinski et al. Adv. Drug Del Rev., 23, 3-25, 1997 

Cons: 

Precipitation (not solubility) 

Less reproducible 

No control of the solid phase 

DMSO interference, however typically 

below 1% 

Interference due to difference in particle 

size 

Interference due to chromophore 

Risk of overinterpretation in later 

development 


Solubility. How to perform Nephlometric solubility 

Nephleomtery -> scattered light 

– Needs special instrument 

Turbidimetry -> transmitted light 

– Std. UV/vis spectrophotometer can be used. 


Aapp or AUC 


Add 1 µl DMSO 

stock solution 

Thermostat 

jacket 

Incubate 

1 Min 



Measure 

Turidity 

A app 

Incubate 

1 Min 

Measure 

Turidity 

A app 



Mag. 

stirrer 

0,2 

0,18 

0,16 

0,14 

0,12 

0,1 

0,08 

0,06 

0,04 

0,02 

0 

0 10 20 30 40 50 60 70 80 

µg/ml 

Lipinski et al. Adv. Drug Del Rev., 23, 3-25, 1997 

Intersection point of bi-linear phases 

at x-axis is the kinetic solubility. 



Alternative method using wellplate technology and robotics 

. . X µl stock 

Cmp 1 

Cmp 2 

Cmp 3 

Cmp 4 

Cmp 5 

Cmp 6 

Cmp 7 

. 

. 

Cmp X 

Neph. Solubility 

Automated data 

processing 

Incubate x min 

Nephlomteric 

plate reader 


Solubility. Potentiometric solubility 

Pros: 

Easy sample preparation 

No standard curve 

Automatic 

Low compound demand (50-1000 µg) 

Thermodynamic solubility 

– With some exceptions 

Limit of detection ~5ng/ml (sup. Info) 

Cons: 

Low throughput 5-10 Cmp per week 

per instrument 

Intrinsic solubility only 

S 0 < ~5 mg/ml 

Technically advanced 

Needs highly skilled personel 

Mathematical fitting 

Limited suppliers: 

pION Inc. (US) 

Sirius Analytical (UK) 

Uncontrolled solid phase 

Works only for ionisable Cmp s 

Requires accurately determined pKa 

Avdeef, A. Pharm Pharmacol. Commun. 1998,4,165-178 


Solubility. Solubility of salts 

Solubility of salts (base): 

pH< pH max solid phase : Salt 

pH> pH max solid phase : Free base 

pH max ~ pKa – 2(3) 

Solubility of monoprotolytic base (pH> pH max ) 

S S o 

S 10 

0 

pka 

pH 

Intrinsic solubility S 0 is defined as the 

solubility of the neutral species 

Solubility of monoprotolytic salt of base is 

defined by the solubility product K sp 

K sp 

 

 

B 

 

Counterion 

Solubility declines at low pH due to common 

ioneffect e.g surplus of Cl - from hydrochloric 

acid. 

Fig. pH solubility of terfenadine depending on counterion 

Reprintetd: W.H Streng et al. J. Pharm Sci. 1984, 73, 

1679-1684 

(Handbook of Pharmaceutical salts. 2002) 


Solubility. What is ”sufficient” solubility 

MAD k 

k 

C 

V 

t 

a 

s 

Si 

Si 

a 

C 

s 

V 

Si 

t 

:First order absorption rate constant 

:Drug solubility 

:Small intestinal volume ~ 250 ml 

Si 

:Small intestine transit time ~ 270 min 

Triad of: 

Potency 

Permeability 

Solubility 

Curatolo, W. Pharm Sci & Tech 1,9, 1998 387-393 

Main barrier is formulation for tox studies: 

Extreme doses >400 mg/kg not unsual > typically 100 s fold higher than future 

human dose. 

Solubility might be too low to reach these extreme doses 

Suspensions could be an approach 

Advanced suspensions e.g Nanomilling for higher exposure per doseunit 


AUC, ng/ml *h 

S, mg/ml (free base) 

Solubility. Exposure screen, an example 

NSXXXX: 

Exposure and solubility of NS XXXX, 5 mg/kg PO in the mouse 

Weak base pKa: 9.5, 

120 

35 

S 0 =0.02 mg/ml 

BCS class I 

Six salts were prepared 

Exposure was evaluated 

Low exposure of HCl most 

likely due to ventricular 

common ion effect 

100 

80 

60 

40 

20 

30 

25 

20 

15 

10 

5 

Acetate salt was selected 0 

0 

Exposure 

Solubility 


AUC, ng/ml * h 

S, mg/ml (free base) 


NSZZZZ: 

Very weak base pKa: 2.16, 

S 0 =0.0005 mg/ml 

Three salts were prepared 

Exposure was evaluated in the 

rat 

Sulphate salt was selected 

Exposure and solubility of NSZZZZ 8 mg/kg PO in the Rat 

4500 

0,20 

4000 

0,18 

3500 

0,16 

3000 

0,14 

0,12 

2500 

0,10 

2000 

0,08 

1500 

0,06 

1000 

0,04 

500 

0,02 

0 

Bromide Sulphate Hydrochloride 

0,00 

Exposure (AUC) 

Solubility 

Aqueous pH ~ 2 


S, mg/ml 

S, mg/ml 


NSYYYY: 

Very weak base pKa: 4.2, 

Structure liable to strong acid (for salt forming) 

Free base was selected 

S 0 ~0.005 mg/ml 

BCS class II 

60 

50 

Solubility of NSYYYY vs pH 

35%HPbCD 

15%MebCD 

100 

10 

Solubility of NSYYYY vs pH 

35%HPbCD 

15%MebCD 

40 

30 

25%HPbCD 

15%HPbCD 

1 

25%HPbCD 

15%HPbCD 

20 

Water 

0,1 

Water 

10 

0,01 

0 

0,001 

-1 0 1 2 3 4 5 6 7 

-1 0 1 2 3 4 5 6 7 

pH 

pH 



Formulation: 

1: 15 % HP-ß-CD in phosphate buffer, pH = 2 (solution) 

2: 15 % HP-ß-CD in tartrate buffer, pH = 3 ( partly susp. Nanomilled) 

3: 0.5 % HPMC in tartrate buffer pH = 3 ( std. suspension. ) 

4: 0.5 % HPMC in tartrate buffer pH = 3 (nano-milled suspension) 

Prepared at 10 mg/ml 

D(0.5) standard suspension: 73 µm 

D(0.5) nanomilled suspension: 0.39 µm 

Conclusions: 

Solution (1) or partial solution (2): AUC↑ 7-8 times 

relative to std. suspension (3) 

Nanomilling (4): AUC ↑ 3-4 times rel. to std. 

Suspension (3) 

Normalisation of ventricular pH in the dog is useful 

(Preferable pH~3) 


Solubility. 

Conclusion 

HTS kinetic solubility screening is useful for in-vitro screening as 

precipitation from DMSO solution is undesired 

Shakeflask solubility useful in QL phase for developing in-vivo dosing 

vehicles. Solid-state analysis of precipitate possible 

Solubility is not the only parameter to consider when selecting solid phase 

Solubility is ONE parameter in a thorough material characterisation 

program 

Melting 

point 

In-vivo 

exposure 

Salt 

stoichiometry 

Hygrosco 

picity 

Absolute 

stucture 

S-XRD 

6 th Annual Improving Solubility for Pharmaceutical and Biologics. 21 st -23 rd June 2010. 19 

Processability 

Solid 

state 

stability 

Synthetic 

yield 

Polymorp 

hism 

Crystallinity

Solubility. 

Acknowledgements 

Department of DMPK, NeuroSearch A/S 

Department of Preformulation, NeuroSearch A/S 

Special thanks to: 

Dr. Anne-Marie Jacobsen, Head of DMPK 

Thomas Jacobsen M.Sc, Research Scientist, DMPK 

Dr. Anne Zimmermann, Preformulation Chemist 

PharmaIQ for hosting this venue

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