1 Drug....Many Complaints!

Louise Rutherford

Paresh Masuria GlaxoSmithKlin

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Agenda

Background Study Information GLP Oral Stability Study

◦ Vehicle Assessment work hERG Solubility Study

◦ Spectroscopy Analysis GLP hERG Study Intravenous Study

◦ Vehicle Assessment work Formulation Analysis Support Conclusion

First study that we ran was a rabbit wedge study with Batch 1◦ This was formulated at 43 µg/mL in 0.1% DMSO in Tyrodes solution ◦ No problems were noticed at the time of preparation and study was completed with no issues

Second study was Dog MTD study which was formulated with Batch 2◦ Simple suspension formulated in 1% (w/v) Aqueous Methylcellulose at a concentrations of 6 and 20 mg/mL, homogenised using an

Ultra-Turrax mixer ◦ Prepared on the day of dosing ◦ No formulation issues were noticed although the drug was not tolerated at 20 mg/mL (100 mg/kg)

Third Study was a Dog CVS study which was formulated with Batch 3◦ Again, as with the MTD study this was a simple suspension formulated in 1% (w/v) Aqueous Methylcellulose at a concentration of 6

mg/mL, homogenised using an Ultra-Turrax mixer◦ Prepared on the day of dosing◦ No formulation issues were noticed

The in-vitro solubility study was started with Batch 4◦ No issues and solubility found to be 303 mg/mL in DMSO◦ No other solubility was started at this stage

The GLP in-vitro stability study in DMSO was conducted with no issues

Background – Early Studies

4 & 14 Day Rat Dose Range Finding Study◦ This was conducted using Batch 4◦ Again prepared on the day of dosing in 1% (w/v) Methylcellulose at concentrations of 0

and 3 mg/mL, homogenised using an Ultra-Turrax mixer – no formulation issues noticed◦ However, whilst this study was ongoing the GLP formulation stability study was also

starting………

GLP Oral Stability Study◦ This was also conducted using Batch 4◦ Concentrations of 0.1, 1 and 20 mg/mL were initially selected as these would cover

maximum concentrations required to support both upcoming Rat and Dog studies◦ Formulated in 1% (w/v) Aqueous Methylcellulose◦ Homogenised using an Ultra-Turrax mixer AND……..

When The Problems Began...Complaint 1

Oh Dear... Batch 4 at 20 mg/mL did not form a suspension

Saw agglomeration / gumming to the Turrax head and the sides of the glass

Impossible to remove from the Turrax head!

Decided to reduce the top concentration down to 10 mg/mL ◦ Prepared a 10 mg/mL and decided to stir magnetically and sonnicate prior to homogenising

using the Ultra-Turrax mixer

◦ Compound ‘gummed’ to the flea and the side of the glass container and again during homogenisation agglomerated on the Turrax head

◦ 0.1 and 1 mg/mL concentrations seemed to have formed a suitable suspension and were therefore sampled and submitted for analysis.

◦ The two higher concentrations were dropped from the study

GLP Oral Stability Study

0.1 mg/mL Day 0 Day 15

Determined Sample Concentration (mg/mL) 0.110 0.108

Nominal Concentration (%) 110 108

Stability Results – 0.1 and 1 mg/mL

Stability of Compound 1 at a Nominal Concentration of 1 mg/mL in 1% (w/v) Aqueous Methylcellulose when Stored at Approximately 4°C, Protected From Light

1 mg/mL Day 0 Day 15

Determined Sample Concentration (mg/mL) 0.981 0.980

Nominal Concentration (%) 98.1 98.0

Stability of Compound 1 at a Nominal Concentration of 0.1 mg/mL in 1% (w/v) Aqueous Methylcellulose when Stored at Approximately 4°C, Protected From Light

0.1 mg/mL Day 0 - Homogeneity Top Middle Bottom

Determined Sample Concentration (mg/mL) 0.109 0.110 0.110

Nominal Concentration (%) 109 110 110

Homogeneity Results

0.1 mg/mL Day 15 Homogeneity Top Middle Bottom

Determined Sample Concentration (mg/mL) 0.107 0.108 0.109

Nominal Concentration (%) 107 108 109

1 mg/mL Day 0 - Homogeneity Top Middle Bottom

Determined Sample Concentration (mg/mL) 0.981 0.985 0.978

Nominal Concentration (%) 98.1 98.5 97.8

1 mg/mL Day 15 - Homogeneity Top Middle BottomDetermined Sample Concentration

(mg/mL) 0.978 0.977 0.986

Nominal Concentration (%) 97.8 97.7 98.6

1% (w/v) Aqueous Methylcellulose was a suitable vehicle for concentrations of 0.1 and 1 mg/mL◦ But not suitable for higher concentrations

With what we had seen in the stability study we decided to review the formulations for the 4/14 day ongoing rat DRF study◦ This was currently being prepared at 3 mg/mL

We examined the remaining formulation being returned after dosing and noticed the same ‘gumming’ / sticking to the bottom of the jar◦ Although this was slight in comparison to the 10 mg/mL

It was decided, however to perform formulation analysis on the next preparation to ensure that this sticking had not compromised the study

Homogeneity analysis was therefore performed

Next Steps:

Top Middle Bottom

Determined Sample Concentration (mg/mL) 3.07 3.08 2.97

% of Nominal Concentration 102 103 98.9

Homogeneity – 3 mg/mL

• All results were within ±5% of the mean and the mean was within ±10% of nominal

Results showed that shortly after preparation formulations are suitable for dosing and therefore this study was not compromised and was run to completion

However, the sticking / gumming appears to happen over time. Therefore, it was thought that this vehicle was not suitable for this batch of material for longer term studies where formulations would be prepared in advance

After discussions with Pharm Dev, we embarked on a vehicle assessment to see if we could determine a new formulation for this compound

Timelines were very tight, a dog DRF study and another rat DRF study were scheduled in for a few days time (early Oct) and project team members wanted to fit in one month studies before Christmas so long delays to these studies were not an option

Due to the toxicity of the compound we would only now require concentration up to 10 mg/mL

Next Steps:

Initially looked at added a surfactant to see if this would help disperse the drug◦ Tween 80 and Sodium Lauryl Sulphate◦ We looked at:

0.1, 0.5 and 1% Tween 80 0.1, 0.2 and 0.5% SLS

The addition of surfactants overall improved the dispersity of the formulation although SLS was much better than Tween 80◦ The addition of Tween 80 was initially good but overtime the formulations aggregated and once again gummed to the side of the

glass

However, formulations were now a solution and not a suspension at 10 mg/mL◦ Required sonnication and stirring prior to homogenising using the Ultra-Turrax mixer

0.1, 0.2 and 0.5% SLS

1% (w/v) Methylcellulose containing 0.2% (v/v) SLS was selected

The assessment was completed in 1 day

Vehicle Assessment

Formulations with SLS...

Spot The Difference?

The main concern from the project team at this point was that the formulation was now a solution and all previous work had been conducted as a suspension◦ Concerns over increased bioavailability – increased exposure

Concerns over this current batch and its potential to gum in the presence of aqueous and how it would behave in-vivo◦ Could the same gum phenomenon occur in the GI tract

Concerns over the suitability of this batch of compound and its physical properties◦ MedChem were due to release a new batch of compound for upcoming IV work and the CD1

batch was due early November

Concerns

The project team decided to go with the new improved formulation with the addition of SLS◦ It was thought that any increase in exposure would be slight and the risk was low◦ The Dog DRF was only delayed by 7 days and the Rat DRF was conducted on time with the new

formulation◦ Both were completed with no issues

The GLP oral stability study was re-started with the new formulation with no analytical issues ◦ All oral studies run to date, including 1 month GLP studies, performed with the new formulation have

been run with no formulation or analytical issues

Next Steps:

As a result of what we had noticed with this batch we went back to look at previous batches as these had been formulated up to 20 mg/mL (Dog MTD) and 6 mg/mL (Dog Pre CS CVS)◦ We did not see the same gumming with Batch 2+3 that we had seen with Batch 4 and higher

concentrations were achievable◦ It was concluded that these studies had not been compromised and this was a batch specific

issue

This however lead to discussion within the project team as to how this and future batches of this compound had been or were to be manufactured

Previous Batches

We started the hERG solubility with Batch 4◦ Maximum solubility in DMSO was 303 mg/mL

◦ Final vehicle for a hERG study is 0.3% DMSO in hERG PSS◦ Therefore a 1 in 333.33333 dilution of the DMSO stock

◦ First dilution was 0.909 mg/mL in 0.3% DMSO in hERG PSS Was not soluble and again saw the same ‘gumming’ The compound formed a ‘gum’ ball

◦ Second dilution we perform is from a stock 1 in 10 of the maximum DMSO solubility figure◦ In this case this was 30.3 mg/mL and therefore gave a final hERG solubility concentration of

0.0909 mg/mL in 0.3% DMSO in hERG PSS Again this was not soluble and saw the same gumming / sticking to glass

hERG solubility...Complaint 2

We went down as low as 0.0909 µg/mL and this still wasn’t soluble and showed the same gumming / sticking to glass as seen with the oral formulation◦ This concentration would be too low to conduct on a hERG study

We decided to perform some UV spec and HPLC analysis on the higher concentrations of 0.909 and 0.0909 mg/mL◦ This was to ascertain what proportion of the compound was in solution and what proportion was gumming

and sticking to glass◦ Also attempted to remove the ‘gum’ ball and dilute with diluent to see what proportion of this was

compound or if this was an impurity◦ This involved developing analytical methods

hERG solubility

Spectroscopy Analysis...Complaint 3

UV-Vis Spec – Phase 1

Analytical support provided within a short time period◦ Labour intensive

Formulation was insoluble at the high concentration (0.909 mg/mL)◦ Residue was formed at the bottom of the container

Result was outside the acceptable criteria of ±10% of nominal◦ Expected considering the residue formation◦ Proved that considerable proportion of Test Material present within final formulation◦ Potentially over 30% of TM may be present within the residue or lost within an impurity

Nominal Conc. (mg/mL) Calculated Conc. (mg/mL) Nominal Conc. (%)

0.909 Test 0.606 66.71

HPLC Analysis – Phase 1 Analysis of samples at both concentrations (0.0909 and 0.909 mg/mL)

The residues formed within formulations of both concentrations were also analysed◦ Gum produced at 0.909 mg/mL◦ Insoluble flake produced at 0.0909 mg/mL◦ Analytical diluent added to residue and shaken to attain homogeneous mix. Resultant mix analysed

All sample results were outside the acceptable limits of ± 10% of nominal◦ Both UV and HPLC analysis confirmed similar results at 0.909 mg/mL (66.71% from UV-Vis, 77.45% from HPLC)◦ The flake from the 0.0909 mg/mL could not be dissolved in the diluent◦ Suggested that this may have been an impurity formed in the hERG PSS rather than compound separating out◦ The residue (gum ball) for the 0.909 mg/mL concentration was easily dissolved in the analytical diluent◦ Suggesting that this was possibly the result of a combination of compound and impurity◦ Results suggested that even though precipitation of the compound was observed, a proportion of the compound was in solution◦ Potentially a way forward for the upcoming hERG study

Nominal Conc. (mg/mL) Calculated Conc. (mg/mL) Nominal Conc. (%)

0.909 Test 0.704 77.45

0.909 Residue 0.112 12.32

0.0909 Test 0.0661 72.72

0.0909 Residue No Test Peak Detected -

UV-Vis Spec – Phase 2 We then prepared hERG solutions at 0.0909 mg/mL with various TM batches

◦ Batch 1, Batch 3 and Batch 4 (the problematic one!)◦ At this conc. batches 1 + 3 appeared to be in solution, although slightly hazy – no sticking noticed◦ Batch 4 precipitated out into a gum

Assessment of hERG formulations once more◦ In order to investigate if the insolubility issue was batch specific◦ Provide information on potential suitable batches of TM that could be used for hERG study

Formulations with batches 1 + 3 were within or close to acceptable criteria of ±10% of nominal◦ Indicative of the hazy solutions produced◦ Low result for batch 4 was indicative of the gummy substance being formed

New GLP batch of Test Material (Batch 5) was ordered in specifically for the IV study

Test Material Batch Nominal Conc. (mg/mL) Calculated Conc. (mg/mL) Nominal Conc. (%)

Batch 1 0.0909 0.087 95.25

Batch 3 0.0909 0.077 84.46

Batch 4 0.0909 0.054 59.86

There appeared to be a big difference in the earlier batches and this problematic Batch 4

The project team Pharm Dev rep confirmed that Batch 4 was manufactured differently to previous batches and that the issues we have seen with Batch 4 may be resolved when the CD1 batch was released.

We supplied Pharm Dev with a detailed description of what we had seen with the different batches in the different formulations and this was fed back to the project team and the chemists

Pharm Dev were trying to understand the impact that synthesis, isolation and residual solvent/water levels have on the ‘developability’ of the material and the issues we had seen and our observations would help tease out the risks with the CD1 batch

It was decided to wait until receipt of the CD1 batch to resume the hERG solubility

hERG solubility

Although the hERG study was completed successfully this was not without its own problems!!

The study was conducted in two phases – phase 2 was carried out due to problems safety pharm had with their cells – both phases had analysis performed on the DMSO stocks and control DMSO

In both phases additional peaks were observed in the analysis of the test stock solutions. ◦ These peaks were only seen in the test stock – they were not seen in the DMSO control sample or the analytical standards◦ As the same bottle of DMSO was used for both control and test formulations and the same diluent was used for both test

and control dilutions, this ruled out contamination in the DMSO and Diluent◦ This peak had also not been observed during analytical method development

After discussions with the analysis group it became apparent that they were using a different batch of the compound for their method development and their analytical standards to what was being used on the hERG study◦ It was thought these peaks could be a result of batch variation – this was therefore investigated

GLP hERG Study...Complaint 4

We supplied the analysis group with the current Batch 6 to make up their standards with

We re-prepared the DMSO test formulation using the batch DMPK had been using (Batch 2) and also re-sampled the DMSO stock that we had seen these additional peaks in◦ The analytical results showed the presence of additional peaks in ALL samples and analytical

standards that had been prepared using Batch 6◦ They were not present in the samples submitted using Batch 2

These results confirmed that the additional peaks seen in the first analysis were a result of a component in Batch 6 that had not been seen in Batch 2 that was used for method validation and preparation of standards and was not a degredent◦ This had no impact on the formulations which were all within ±10% of nominal

hERG Study – Additional peaks

Batch 6 was received and hERG solubility was resumed

No gumming / sticking was seen with this batch this was very similar to earlier batches

A concentration of 129.2 µg/mL was achieved in 0.3% DMSO in hERG PSS◦ 1421 times higher than Batch 4

The GLP hERG study was carried out successfully with Batch 6

hERG solubility

Pharm Dev provided vehicle of 5:20:75 (v/v/v) Ethanol:Propylene Glycol:10% (w/v) Aq. 2-Hydroxypropyl β-Cyclodextrin

To support 3 Hr rat, and 1 Hr dog infusion studies

Vehicle used in rats for bolus injections

Each component individually used by infusion administration

Vehicle control only administered to rats during the study◦ Resulted in bruised / black tails◦ Vehicle deemed not suitable for Test Article dosing◦ Problematic components likely to be Propylene Glycol and Cyclodextrin

Intravenous Formulation...Complaint 5

Needed to identify a new formulation within 1-2 weeks!

Dispensary accepted challenge

Intravenous Vehicle Assessment

Ethanol > 200 mg/mL DMSO – 303 mg/mL Solutol – 50 mg/ml Cremophor > 200 mg/mL Propylene Glycol > 200 mg/mL PEG-400 > 200 mg/mL Saline < 1 mg/mL 10% (w/v) Aq. Captisol in Saline < 1 mg/mL

Solubility Screen

PD Vehicle: 5:20:75 (v/v/v) Ethanol:PG:10% (w/v) Aq. 2-HPBC

Vehicle Assessment 1 5:20:75 (v/v/v) Ethanol:PG:10% (w/v) Aq. Captisol in Saline – 10 mg/mL in solution

◦ Substituting β-Cyclodextrin for Captisol

5:20:75 (v/v/v) DMSO:PEG-400:10% (w/v) Aq. Captisol in Saline – 10 mg/mL in solution ◦ Substituting all 3 components to achieve same solution

25:75 (v/v) PEG-400:10% (w/v) Aq. Captisol in Saline – 10 mg/mL in solution◦ Could not be administered to rats, due to issues concerning the time of infusion

5:5:90 (v/v/v) DMSO:Cremophor:10% (w/v) Aq. Captisol in Saline – 10 mg/mL in solution ◦ Surfactant was very good at solubilising TM, as seen with the oral work

5:5:90 (v/v/v) DMSO:Cremophor:10% (w/v) Aq. Captisol in Saline – 10 mg/mL in solution ◦ Scale up preparation

5:5:90 (v/v/v) DMSO:Solutol:10% (w/v) Aq. Captisol in Saline – 10 mg/mL in solution ◦ Substituting Cremophor for a more favourable surfactant

PD Vehicle: 5:20:75 (v/v/v) Ethanol:PG:10% (w/v) Aq. 2-HPBC

Vehicle Assessment 2 5:95 (v/v) DMSO:10%(w/v) Aq. Captisol in Saline – 10 mg/mL

◦ No solution – clumping occurs 5:95 (v/v) Solutol:10% (w/v) Aq. Captisol in Saline – 10 mg/mL

◦ In solution

Vehicle Assessment 3 5:95 (v/v) Solutol:10% Saline – 10 mg/mL

◦ In solution. Removal of Captisol 2.5:97.5 (v/v) Solutol:Saline – 10 mg/mL

◦ Not in solution on cooling 3.75:96.25 (v/v) Solutol:Saline – 10 mg/mL

◦ In solution 3:97 (v/v) Solutol:Saline – 10 mg/mL

◦ Not in solution on cooling 5:5:90 (v/v/v) DMSO:Solutol:Saline – 20 mg/mL

◦ In solution

Hmmm.......

PD Vehicle: 5:20:75 (v/v/v) Ethanol:PG:10% (w/v) Aq. 2-HPBC

Final vehicle: 5:5:90 (v/v/v) DMSO:Solutol:Saline selected◦ Reduction in overall solvent content compared to the original vehicle◦ 3:97 Solutol:Saline could be dosed at 5 mL/Kg/Hr [50 mg/Kg/Hr]◦ 5:5:90 (v/v/v) DMSO:Solutol:Saline can be dosed at 2.5 mL/Kg/Hr [50 mg/Kg/Hr] ◦ Has lower total Solutol content

7 Day, 3 hour rat infusion study (vehicle only) run without effects

Solutol cannot be administered IV in dog due to sensitivity reactions

Further IV vehicle assessment required to provide separate vehicle for dog study!◦ Dog study required lower concentrations◦ Vehicle only needs to be tolerated for 1 Hr infusion

Not Quite There...Complaint 6

Original vehicle provided by PD: 5:20:75 (v/v/v) Ethanol:PG:10% (w/v) Aq. 2-HPBC may have been tolerated in the dog

25:75 (v/v) PEG-400:10% (w/v) Aq. Captisol in Saline considered most appropriate◦ Identified during earlier vehicle assessment work ◦ At a lower dose volume (2 mL/Kg)◦ This vehicle could not be used for the rat study, due to sensitivity issues caused by 3 hours infusion

Caused scale up issues◦ Solution appeared slightly hazy prior to filtration◦ May have potentially affected TM conc. within formulation

Performed analytical work to develop method for verification of the filtrate suitability◦ Time consuming and labour intensive task

Vehicle Assessment 4

Spectroscopy Analysis...Complaint 7

HPLC Analysis – Phase 2 Samples were analysed at concentrations of 10 and 16 mg/mL (both filtered and unfiltered)

◦ Intended concentrations likely to be dosed during the IV study

Samples were analysed on Days 0 and 6 for assessment of stability over storage at approximately 4ºC, protected from light

Day Number Sample Nominal Conc. (mg/mL)

Calculated Conc. (mg/mL) Nominal Conc. (%)

0 Unfiltered 16 13.704 85.65

0 Filtered 16 13.311 83.19

0 Unfiltered 10 8.809 88.10

0 Filtered 10 9.296 92.96

Day Number Sample Nominal Conc. (mg/mL)

Calculated Conc. (mg/mL) Nominal Conc. (%)

6 Unfiltered 16 13.759 85.99

6 Filtered 16 13.881 86.76

6 Unfiltered 10 9.151 91.51

6 Filtered 10 9.272 92.72

All results were close to or within acceptable limits of ±10% of nominal concentration◦ Ranging from 83.19% - 92.97%

Filtration had no effect on the sample concentration◦ Only a single reduction in concentration observed for Day 0 – 16 mg/mL sample (2.46%)

No significant loss in concentration at both levels from Day 0 to Day 6◦ Only a single reduction in concentration observed for 10 mg/mL – Filtered sample (0.24%)

Stability proven for 6 days after storage at approximately 4ºC, protected from light◦ To support the scheduled dog infusion study

Formulation proven to be suitable!

Summary of Analysis Results

Intravenous programme terminated 1 day before first animal received the drug in either vehicle!

Conclusion 7 complaints raised from 1 Drug related to 9 different studies in just over 5 weeks

Oral Studies;◦ 1% Methylcellulose was not a suitable oral vehicle for higher concentrations◦ Caused gumming / sticking to the glass◦ Addition of SLS improved the dispersity of the compound to produce a solution

hERG Solubility;◦ The issue with the hERG solubility was batch specific◦ Batch 4 was not suitable for hERG formulations. GLP hERG study was successfully completed with Batch 6◦ The additional peaks seen in the hERG study were also a batch specific issue (Batch 6) and were not a result of contamination or a degradant

IV Studies;◦ 4 different IV vehicle assessments performed to determine optimum vehicles for both rat and dog infusion studies◦ 2 individual vehicles identified◦ Separate vehicle required for dog study, due to sensitivity reactions in the animals◦ Formulation analysis work performed to resolve the scale-up issues observed with this vehicle◦ Verified that filtration of the dog formulation did not affect the TM conc.

Very labour intensive work all completed within stringent timelines◦ No impact on any studies◦ All completed without any compromise◦ Over 100 hours of work involving 6 different people completed for the big go-ahead.....

Just another day in Dispensary