Excipients and impurities in non clinical formulations...
Transcript of Excipients and impurities in non clinical formulations...
Excipients and impurities in non clinical formulations: challenges for data interpretation and importance of experience
Annecy, October 17th, 2014 Serena Cinelli
Highlights
Case study 1 (impurities): toxic effects detected in genetic toxicity testing
Case study 2 (impurities): toxic effects detected in general toxicity
testing Case study 3 (excipients): toxic effects detected in general toxicity
testing
Starting Materials
Intermediates
Solvents
Catalysts
«Substance» «Final Product»
Reagents in excess By-products
Starting materials
Reagents in excess By-products
Degradation products
Introduction
Research Development Launch
Lead optimization
Lead discovery
Pre-clinical development
Clinical development Registration
Identification and qualification of impurities
Marketing
Pharmaceuticals
Case study 1
Regulatory Ames test (5 tester strains)
NCE
Purity > 99%
No structural alerts
Chemical structure
Alert
Elaboration
0
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0,00 1,00 2,00 3,00 4,00 5,00 6,00
Dose level (mg/plate)
Reve
rtant
s/pl
ate
TA1535 TA100
Statistically and biologically significant increases were observed with TA1535 and TA100 which are predominantly sensitive to base pair mutagens
Results - Case study 1
Result interpretation - Case study 1
Based on our experience, it was considered unlikely that similar chemical structure could cause this mutagenic effect
Sponsor was involved in an extensive discussion
Production process was examined (source of possible contaminants and nature of possible impurities) Identification of NaN3 in one production step → suspect of contamination in the drug substance
Problem solution - Case study 1
The number of revertant colonies at 5000 µg/plate was comparable to that obtained at 0.5 µg NaN3/plate Thus the level of NaN3 would correspond to a 0.1‰ concentration in the API At this level the NaN3 was not detectable by analytical methods
The Sponsor worked on the specific production step to eliminate
NaN3
Subsequent lots of drug substance were examined by Ames test to assess the residual presence of NaN3
which finally turned negative
Case study 2: ADA testing completes the comparison exercise of biosimilars
Study design:
• Preclinical development of a biosimilar protein
• Rat was a relevant model
• Analytical methods supported similarity
• 4 week subcutaneous toxicity study with TK and
ADA measurement
Preclinical immunogenicity: Why?
Careful antibody monitoring throughout the preclinical development is important: to ensure that the animals were exposed to the expected level
of product to confirm similarity in case of process change
Factors which affect immunogenic potential of a biotech product
•Dose •Frequency •Route of administration •Impurities •Aggregates
Males Day 1
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0 4 8 12 16 20 24Time (hr)
Item
Con
cent
ratio
n (n
g/m
l)
8M- Low Test 9M- High Test 10M- High Ref.
Case 2 -Toxicokinetics in Study A
Males High doses
0
500
1000
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0 4 8 12 16 20 24Time (hr)
Item
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cent
ratio
n (n
g/m
l)
9M- D1 - HiTest 10M- D1 - HiReference 9M- 4W - HiTest 10M- 4W - HiReference
Case 2 -Toxicokinetics in Study A
End of TreatmentMales
-0.100
0.400
0.900
1.400
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OD
nor
mal
ized
Control Low test High Ref.High Test
Case 2 – ADA in Study A
-100
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0 120 240 360 480 600
Item
Con
cent
ratio
n (n
g/m
l)
Time (min)
Females Day 1
8F- Low Test 9F- High Test 10F- High Ref.
Case 2 -Toxicokinetics in Study B
Females High doses
-100
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0 120 240 360 480 600Time (min)
Item
Con
cent
ratio
n (n
g/m
l)
9F- D1 - HiTest 10F- D1 - HiReference9F- 4W - HiTest 10F- 4W - HiReference
Case 2 -Toxicokinetics in Study B
End of TreatmentFemales
-0.1
0.4
0.9
1.4
1.9
2.4
2.9
OD
nor
mal
ized
Control Low test High Ref.High Test
Case 2 – ADA in Study B
Photomicrograph of the subcutaneous injection site from one high dose animal. Note: Perivascular mononuclear cell infiltration (i.e., lymphocytes and plasma cells), surrounding two blood vessels.
Case 2 – Histopathology in Study B
• RTC: Aggregates? • Analysis of final formulation by more accurate systems • Better characterization confirmed the initial
hypothesis • Aggregation occurred during storage and handling
conditions (standard for a preclinical laboratory) • These conditions would be most probably applied in
the hospital as well, thus posing a problem of clinical immunogenicity
Case 2 – Investigation
Case 3 – Juvenile study
Study design Slow intravenous administration in juvenile rats Treatment started at Day 21 of age (pre-puberty stage) Treatment lasted for 4 weeks (up to Day 49 of age) Recovery period: 28 days All standard observations of a 4 week study (CS,BW,FC, Clinical pathology, OW, Macro and Micro) Additional tests:
• Vaginal opening • Testis descent and preputial separation • Motor activity • Water-filled Y-Maze • Accelerating rota-rod
Toxicokinetics
•Day 28 of treatment: blood samples from 6 animals/sex/group
•Time points: 3 min, 15 min, 30 min, 1 hour, 4 hours
•Maximum of 3 alternating time points per animal
•Results: all animals exposed
no evidence of gender differences
rapid kinetic and elimination
Case 3 – Juvenile study
Study data
• Body weight, General health and Learning performance no
change in either gender
• Clinical pathology, Haematology and Histopathology dose-
related findings in treated males and females (changes
associated to the test item MoA)
• Male treated rats delay in preputial separation
• Female treated rats no change in vaginal opening time
Case 3 – Juvenile study
Vehicle data from previous studies
Vehicle Low dose Medium dose High dose
Case 3 – Juvenile study
30
40
50
* * *
38 39 43 43 42
WHY? ALL treated males WITHOUT dose relation?
Test item or formulation?
Investigation • Both estrogenic and anti-androgenic chemicals may induce
delays in male puberty • The test item was well characterized and known to lack of any
hormonal activity • Certificate of analysis reported presence of Parabens, as
preservative in the final formulation • Some scientists reported that parabens exert anti-androgenic
activity • Suggestion to investigate the “role” of the formulation
composition
An additional study with a new formulation was proposed
Case 3 – Juvenile study
Case 3 – Juvenile study
New supportive study
• New formulation (no parabens)
• Intravenous administration in male rats (from Day 21 of age up to
Day 49)
• 2 groups (control and high dose)
• Results: Preputial separation was unaffected by treatment
Conclusion
Importance of the CRO experience on data interpretation
Positive results
Deep analysis of experimental settings and, through a fruitful collaboration with the Sponsor, recognition of the nature of the observed toxicity, the cause of concern and its origin
Annecy, October 17th, 2014 Serena Cinelli
Merci pour votre attention