1. The Value of Digital Photography By Louise Rutherford and Dean Hatt

2. Agenda – Introduction – Case Study A – Case Study B – Case Study C – Case Study D – Case Study E – Conclusions – Questions Presentation title2

3. Introduction – Digital photography was introduced into the Dispensary in 2008 to primarily assess potential colour changes on GLP DMSO stability studies – Over the last 6 years we have managed to capture digital images on over 150 stability studies – Approximately 20% of these have demonstrated a colour change over time – The set up and use of digital photography on stability studies has previously been presented to the CHAOTIC group – The use of digital photography has now been extended beyond stability assessments Presentation title3

4. Case Study A – Compound A was tested within Pharmaceutical Development on a small scale and shown to have high aqueous solubility – A formulation in saline was required for continuous infusion to support in-vivo studies – Dispensary were responsible for the development of in-vitro formulations in DMSO/Water/hERG PSS – Initiated a solubility assessment on an early batch of compound Solubility Assessment Presentation title4

5. Compound A – Early Batch Solubility in Water Presentation title5

6. Compound A – Formulations in Saline Presentation title6 Compound A. Batch Colour Differences –Early Batch –New Batch

7. Compound A – To summarise: – Problem 1 – Formulation changing form over time producing ‘worm cast’ – Problem 2 – Dramatic colour difference between batches – Problem 3 – Extraordinary differences in formulations of the SAME batch prepared in the same conditions but using compound from different containers Presentation title7 Multiple Problems Pharmaceutical Development Container – New Batch Dispensary Container – New Batch

8. Case Study A – Pictures were sent to the project team which immediately allowed escalation of the issue and meetings were set up to discuss. – Lab investigations started in collaboration between SA and Pharmaceutical Development – Likelihood this is related to the formation of a trihydrate – Continuous infusion studies were delayed as a result of the finding – Formulation work is ongoing Conclusions – Where Are We Now Presentation title8

9. Case Study B – DRF’s had successfully been completed on early batches – Standard suspension in 1% MC at reasonably low concentration – up to 10 mg/mL. – GLP batch was received and stability study was initiated Oral Stability Study Presentation title9

10. Compound B Presentation title10 ‘ Gumming’

11. Compound B – Formulation was stable at up to 1 mg/mL in 1% MC – Formulation not suitable at 10 mg/mL – In-vivo studies potentially required up to 10 mg/mL – SA dispensary conducted a vehicle assessment study – Use of surfactants and intermittent sonnication/stirring were identified as the likely route to take – The addition of 0.2% SLS was selected with the following result........ Presentation title11 Unsuitable vehicle

12. Case Study B – Digital photography was utilised to demonstrate the formulation issue to both SA project team member and Pharmaceutical Development – Agreed that the formulation was unsuitable – This was taken forward to successful stability study with this new vehicle – Both stable and homogenous – This was successfully used in GLP studies – This has also been used with a new salt form of the same drug and this information was also used in developing a further formulation for MNT study Conclusion Presentation title12

13. Case Study C – Stability studies in Water, hERG PSS and an aqueous buffer were successfully conducted up to 100 mg/mL – Solubility assessment demonstrated up to approx 400 mg/mL in all three vehicles – Conducted on an early DRF batch that was made to GxP – All GLP studies scheduled to start at CRO including one month nebulised inhalation studies – A haemolysis assay was conducted in house using the new GLP batch Starting GLP studies Presentation title13

14. Compound C 5 mg/mL in Buffer Vehicle Presentation title14

15. Compound C 12 mg/mL in Buffer Vehicle Presentation title15

16. Compound C New Batch v Old BatchNew Batch – Water V DMSO Presentation title16

17. Compound C Presentation title17 50 mg/mL in buffer vehicle –Dark Deposits on glass

18. Case Study C – Digital photography was able to demonstrate a clear difference in solubility of the new batch – < 5 mg/mL compared with approx 400 mg/mL – Dramatic colour difference between the two batches – Pictures clearly showed the appearance of a dark red substance throughout the formulations – Meetings were held between SA and Pharmaceutical Development – Samples of all formulations sent to Pharmaceutical Development for analysis – Pharm Dev could not identify the dark red substance but analysis results were close to nominal – CRO studies were postponed to allow clean up and formulation re-work to be performed – This was high profile as it was one of the first outsourced packages using the new SA strategy. Conclusions Presentation title18

19. Case Study D – Carcinogenicity study at CRO about to begin – Formulation in 0.5% MC – Compound was a sodium salt – pH was at the top end of the acceptable range pH 12 at 200 mg/mL – Potential reverse solubility but the dark colours made this difficult to assess – Trial formulations at the CRO demonstrated poor physical stability and crystallisation of formulations which failed to re-suspend – SA Dispensary conducted a further stability study looking at the physical stability and how we could advise the CRO on how they could prepare it Physical Instability Presentation title19

20. Compound D 200 mg/mL Day 020 mg/mL Day 0 Presentation title20 Reverse Solubility

21. Compound D Day 7 200 mg/mL not stirredDay 7 60 mg/mL not stirred Presentation title21 Colour Differences

22. Compound D 20 mg/mL60 mg/mL Presentation title22 Day 15 stirred v non stirred

23. Compound D 200 mg/mL Presentation title23 Stirred v Non Stirred

24. Case Study D – Digital photography was used to demonstrate a time course of physical stability at a range of concentrations and the effect of continuous stirring – Crystallisation was not seen on the scale performed in this stability study – Project team were happy for us to make recommendations as to the frequency and scale / detail of preparation to allow consistent formulations on study – At 200 mg/mL the recommendation was to prepare the day prior to dosing and store without continuous stirring – At concentrations between 0.1 and 60 mg/mL the recommendation was to prepare at least 3 days prior to dosing to ensure that a suspension was dose Conclusions Presentation title24

25. Case Study E – Current formulation contained 2-hydroxypropyl beta cyclodextrin – FDA rejected CAC proposal for use in a 2 year carc study due to link with cyclodextrins and pancreatic tumours in rats – Had low and variable exposure – SA conducted a vehicle assessment study – Vehicle selected was 50:50 (v/v) PEG 400: Saline – Formulation a solution – Solution at high levels potentially required heat to maintain – Stability study conducted to assess chemical and physical stability Temperature Effect on Oral Stability Study Presentation title25

26. Compound E Presentation title26 Temperature Related Colour Change –10 mg/mL Day 0 –10 mg/mL Day 7 37°C –10 mg/mL Day 15 37°C –10 mg/mL Day 15 4°C

27. Compound E Presentation title27 –Day 0 100 mg/mL –Day 7 100 mg/mL 37°C –Day 15 100 mg/mL 37°C –Day 15 100 mg/mL 4°C

28. Stability Results Day 0Day 7 (approx 37°C) Day 15 (approx 37°C) Day 15 (approx 4°C) Determined Sample Concentration (mg/mL) 9.04 1 8.728.71 1 9.17 1 % of Nominal Concentration90.487.287.191.7 Presentation title28 –10 mg/mL Stability Results Day 0Day 7 (approx 37°C) Day 15 (approx 37°C) Day 15 (approx 4°C) Determined Sample Concentration (mg/mL) 94.6 1 98.998.5 1 98.7 1 % of Nominal Concentration94.698.998.598.7 –200 mg/mL Stability Results

29. Compound E Day 0 0.1 mg/mLDay 15 0.1 mg/mL – 37°C Presentation title29 Day 0Day 7 (approx 37°C) Day 15 (approx 37°C) Day 15 (approx 4°C) Determined Sample Concentration (mg/mL) 0.1 1 0.0943 0.0832 1 0.0986 1 % of Nominal Concentration 10094.3 83.2 98.6

30. Case Study E – Formulation provided increased and non variable exposure in in-vivo studies – Formulations were chemically stable for up to 15 days at 0.1-100 mg/mL when stored at approximately 4°C – At 100 mg/mL the formulation failed to stay in solution at both tested temperatures, but was re-solubilised when minimal heat applied and/or maintained – Formulations were chemically stable for up to 15 days at 10-100 mg/mL when stored at approximately 37°C but unstable at 0.1 mg/mL at this storage condition – Digital photography was used to assess colour change throughout all phases – Colour changes at 10 and 100 mg/mL and chemical instability at 0.1 mg/mL following storage at 37°C, preclude maintenance of these solutions at this temperature – Solutions at 100 mg/mL to be stored at 4°C and re-solubilised for dosing period only Conclusions Presentation title30

31. Overall Conclusions – Digital photography can be used beyond the original remit of assessing instability – It is a useful tool for assessing: – Batch variation – Insolubility – Physical instability – It can therefore aid in: – Proactive formulation assessment – Reactive formulation issues – Fast, non subjective communication of formulation issues to the relevant people – This is of greater importance with the SA strategy of outsourcing GLP studies where formulations are prepared at a greater scale and on batches manufactured using different synthetic routes Presentation title31

32. Questions?