1. Dean R Hatt

3. Agenda  1. Publications  2. Osmolality – A further insight  3. You say homogeneity, I say homogeneity

4. Publications

9.  Just a shame there is no mention of Chaotic !, without which, the link would not have been made.

10. Osmolality

11. Osmolality - Definition  The concentration of osmotically active particles in solution expressed as osmoles of solute per Kg of solvent  The ratio of solutes (such as electrolytes) to fluid. It is measured in osmoles per kilogram of fluid. One osmole is equal to the molecular weight of the substance in grams divided by the number of ions or other particles the substance dissociates into when in solution.

12. In English......  Its a measurement of solutes which effects tonicity.  Isotonic  Same Number of Solutes as Blood.  High tonicity (hyper)  Leads to water leaving cells – shrinkage and crenation  Low tonicity (hypo)  Leads to water entering cells – cell swelling + rupture

13. Effect on Blood Cells

14. Osmolality – Assessment  All osmolality results of aqueous systems demonstrate linear relationships  All osmolality results of combinations of aqueous systems  ‘broadly’ demonstrate linear relationships  are predictable from individual values  All osmolality results of solvent/aqueous systems are broadly linear  DMSO/Ethanol values are very high  Are these values a true indicator of tonicity ?  Osmolality values of combinations are approx. that of the sum of components  Osmo 1+2+3 = Σ Osmo 1,2,3...

15. Osmolality – Assessment (cont’d)  Osmolality Values of multi-vehicles  can be predicted from osmolality of individual components  Reduce future measurement  Osmolality Values of compound solutions  Can be predicted due to linear relationship  Reduce future measurement  Cannot be predicted by Mwt

16. Osmolality – Assessment (cont’d)  Hypertonic – causes crenation  Can be tested by osmolality but not from haemolysis assay (unless cell death occurs)  Leads to ↑ Na+ in urine, ↑ K+ urine, ↑ Water Intake, ↑ Urine volume  Hypotonic – causes cell lysis  Can be tested by osmolality and haemolysis assay  Leads to ↑ Haem in plasma/urine, Reverse of above findings  We can always add solutes to make more isotonic  Solvent osmolality is likely a red herring and we should consider measuring vehicle – solvent  Haemolysis Assay should be conducted  After assessment of Hypotonicity  Using appropriate dose volume appropriate to dsoing regime

17. A little bit of Research....  Osmolality measurements include both effective (active) osmoles and ineffective (inactive) osmoles  The true osmotic effect is the effective (active) osmolality, which is the tonicity  But, we cannot measure only the active osmoles (tonicity) !!!!

18. So What do we do ?  We need to understand what are active osmoles and inactive osmoles  A quick exercise with some examples

19. Example 1 – Solutions containing solvents  2% (v/v) DMSO in sterile water  Osmolality = 299 mOsm/kg  Isotonic ?  Effective Osmolality = 0 mOsm/kg  Hypotonic  Results in haemolysis  Measure Osmolality of vehicle (without solvent) would be better prediction

20. Example 2 – Solutions containing solvents  5% (v/v) DMSO in saline  Osmolality = 1094 mOsm/kg  Hypertonic ?  Effective Osmolality = 297 mOsm/kg  Isotonic  Does not cause any adverse effects  Measure Osmolality of vehicle (without solvent) would be better prediction

21. Example 3 – Solutions of high drug concentrations  50 mg/mL Drug A in Saline  Osmolality = 1745 mOsm/kg  Hypertonic ?  Effective Osmolality = 297 mOsm/kg  Isotonic  Does not cause any effects  Drug A is not osmotically active

22. Example 4 – Solutions of high drug concentrations  50 mg/mL Drug B in Saline  Osmolality = 1850 mOsm/kg  Hypertonic ?  Effective Osmolality = 297 mOsm/kg  Isotonic  But it causes haemolysis  Drug B is not osmotically active but has direct irritant effect on red blood cells  Haemolysis assay would show this

23. Example 5 – Solutions containing glucose  10% 2-H B Cyclodextrin in Sterile Water  Osmolality = 83 mOsm/kg  Hypotonic ?  So what could we do to improve this ?  Prepare in Saline  10% 2-H B Cyclodextrin in 0.7% Saline  Osmolality = 301 mOsm/kg  Isotonic

24. Example 6 – Solutions containing glucose  10% 2-H B Cyclodextrin in Sterile Water  Osmolality = 83 mOsm/kg  Hypotonic ?  So what could we do to improve this ?  Add Glucose ?  10% 2-H B Cyclodextrin in 0.4% aq. Glucose  Osmolality = 299 mOsm/kg  Isotonic ?  Glucose is rapidly metabolised, so effective osmolality is that of 10% 2-H B Cyclodextrin in Sterile Water  Hypotonic = Haemolysis

25. Summary  Effective Osmolality (isotonicity) = Osmolality - Ineffective Osmolality  Consider measuring osmolality of vehicle without solvent content  Drug likely contributes to ‘ineffective Osmolality’ so measure osmolality of vehicle alone  Conduct haemolysis assay to determine direct effect of drug on red blood cells  Be careful with glucose – preferential metabolism

26. Homogeneity

27. Homogeneity - Definition  homogeneity - the quality of being of uniform throughout in composition or structure uniformity - a condition in which everything is regular and unvarying uniformity  ho·mog·e·ny (h-mj-n, h-) n. pl. ho·mog·e·nies Similarity of structure between organs or parts, possibly of dissimilar function, that are related by common descent.  A uniform suspension is therefore homogenious rather than homogenous

28. Homogeneity - Criteria  Each value within 5% of mean  Some companies or formulation types may extend to 10%  Sometimes defined in terms of standard deviation

29. Let us take 3 x 5 mL samples of a 1 mg/mL suspension 0.99 mg/mL 0.98 mg/mL 1.02 mg/mL Homogenious Mean = 1.00 mg/mL

30. Let us take 3 x 2 mL samples of the same 1 mg/mL suspension 0.98 mg/mL 0.96 mg/mL 1.05 mg/mL Homogenious Mean = 1.00 mg/mL

31. Let us take 3 x 0.5 mL samples of the same 1 mg/mL suspension 0.96 mg/mL 0.95 mg/mL 1.05 mg/mL Non -Homogenious Mean = 0.99 mg/mL

32. So What does this mean?  If we take a large sample we have a greater chance of proving homogeneity  Most companies take a standard size (1-5 mLs)  What is the appropriate sample size to take ?  A sample size should be < or = to average dose volume  so we have confidence that each animal receives the correct dose  2-5 mL (rat)  20-50 mL (dog)  0.2-0.5 mL (mouse)  Ideally, we should test down to 0.2-0.5 mL level !

33. How far can you go?  CBAG (capsule based aerosol generator)  creates an aerosol by passing air through a pierced capsule  capsules are filled with 1 – 5 mg of drug / drug blend  the blend may be from 0.01 – 40%  the blend quantity may be from 1 – 100g  A 1 mg weighing of a 0.01% blend would contain 100 ng drug  10% criteria for homogeneity, would require accuracy to +/- 10 ng drug

34. What are the considerations ?  How can we make the blend ?  How can we analyse the blend ?  How can we sample the blend ?  To what accuracy can each phase be done ?

35. What are the considerations ?  How can we make the blend ?  for small quantities consider Retsch mill  serial dilution with small steps  How can we analyse the blend ?  very accurate standards  very low LLQ  How can we sample the blend ?  test and rest for static  vary sample size to assess homogeneity

36. How have we achieved this  To be continued.......