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

50

100

150

200

250

300

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

0

500

1000

1500

2000

2500

3000

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

1500

2000

2500

0 4 8 12 16 20 24Time (hr)

Item

Con

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

1.900

2.400

OD

nor

mal

ized

Control Low test High Ref.High Test

Case 2 – ADA in Study A

-100

100

300

500

700

900

1100

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

100

300

500

700

900

1100

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