How are the Possible Effects of Paternal Drug Exposure on ...

<strong>How</strong> <strong>are</strong> <strong>the</strong> <strong>Possible</strong> <strong>Effects</strong> <strong>of</strong> <strong>Paternal</strong> <strong>Drug</strong>
<strong>Exposure</strong> on Pregnancy Outcome Addressed
during <strong>Drug</strong> Development?
Teratology Society
San Diego, June 2011
Jane Stewart MRCVS PhD
AstraZeneca
Pharmaceuticals
On behalf <strong>of</strong> HESI-DART
ILSI Health and
Environmental Sciences
Institute

Setting <strong>the</strong> scene:
Content
•Pre clinical trial package
•Rodent mating study
options
•Clinical trials sequelae
•Areas <strong>of</strong> uncertainty
Disclosure
The presenter works for AstraZeneca
Pharmaceuticals which may pose a conflict <strong>of</strong>
interest. The original research in this
presentation was funded by AstraZeneca
ILSI Health and
Environmental Sciences
Institute

Pre First Time in Man: Genetic Toxicity Tests
Bacterial Reverse
Mutation (Ames) Test
Bacteria (2/5 strains) removed from
freezer and incubated shaking (37 o C)
overnight
Serial dilution <strong>of</strong> test
compound
INCUBATOR
&
+
/
-
Figure 1
S
9
Metabolic activation system
(Rat liver S9 fraction and c<strong>of</strong>actors)
• Detect :
• base pair substitutions, frameshifts,
transitions/transversions, small
deletions, strand breaks, point
mutations, clastogens & aneugens
In Vitro Micronucleus assay
An
eu
plo
idy
Plates
incubated at
37 o C for 3 days
In vitro Mouse
Lymphoma TK Assay
In vivo Bone Marrow
Micronucleus Test
+ve ?
Micronucle
ated
mononucle
ate cell
Micron
ucleat
ed
binucl
eate
cell
Nucleus
Micronucleus
Cells dispensed
at 1x10 7 /tube (3
hr)
+
/
-
Serial dilution
<strong>of</strong> test
compound
prep<strong>are</strong>d &
cultures treated
Incubated for -/+ 12 S9 days.
Wells containing small &
large mutant clones scored
S
9
CO 2 INCUBATOR
Cultures
incubated at
37 o C for 3
hours
2000 cells/well for mutant
frequency determination
TFT
Cultures
washed,diluted to
2x10 5 /mL&
incubated 24 hr.
Cultures counted,
subbed to
1.5x10 5 /mL&
incubated for
ano<strong>the</strong>r 24 hr
Rats/mice dosed with
compound, three concs, seven
animals / group. Animals
sacrificed 24 or 48 hours later
Micronucleus
Micronuclei may be formed by loss <strong>of</strong> whole
chromosome during division or by chromosome
breakage. The erythrocyte’s nucleus is extruded leaving
any micronuclei behind
Healthy Volunteers v patients
Mutant frequency
calculated
(number mutants
per 10 -6 viable
cells)
Incubated for 8 days.
Wells containing
viable clones scored
Cultures
counted and
plated into
selective (with
TFT) & nonselective
medium
1.6 cells/well for cloning
efficiency determination
2000 cells analysed per animal, number
<strong>of</strong> micronucleated immature
erythrocytes scored
• s
Bone marrow cells spread onto
slides. Slides fixed and stained
(acridine orange)
Femurs removed
and bone
marrow aspirated
Informed consent, & advice
re condom use & procreation

Pre FTiM: General Toxicity Tests
ICH M3: Two species, minimum 2 weeks
Sexually mature animals – usually dog & rat – + recovery groups (preferably)
Organ weights & histopathology (read in a stage-aw<strong>are</strong> manner)

ICH M3: Why <strong>are</strong> clinical trials in men
permitted prior to completion <strong>of</strong> reprotox package?
Histopathology is paramount!
Takayama S et al (1995) : A collaborative study in Japan on optimal treatment period and
parameters for detection <strong>of</strong> male fertility disorders induced by drugs in rats. J. Am.Coll. Toxicol. 14(4)
(1995), 266-292.
Ulbrich, B.; Palmer, A.K. (1995), : Detection <strong>of</strong> effects on male reproduction - A literature survey. J.
Am. Coll. Toxicol. 14(4) 293-327.
Sakai T et al (2000): Collaborative work to evaluate toxicity on male reproductive organs by 2-week
repeated dose toxicity studies in rats. Overview <strong>of</strong> <strong>the</strong> studies, J. Tox, Sci. 25, 1-21
Mangelsdorf I & Buschmann J (2002) Extrapolation from Results
<strong>of</strong> Animal Studies to Humans for <strong>the</strong> Endpoint Male Fertility
German Federal Institute for Occupational Safety and Health
Any as yet undetected adverse effect assumed to be reversible
and manageable via request to avoid procreation?
Adverse effect in mating study assumed unlikely?

Reliance on Histopath for FTiM:
Is <strong>the</strong>re a problem?
HESI DART survey – 16 pharma
• Sasaki et al BDR (Submitted) “Incidence & nature <strong>of</strong> testicular
toxicity findings in pharmaceutical development”
• Sexually mature animals – common but not universal
• 2 examples <strong>of</strong> testes lesions 1st detected in studies > 1 month
• 2 nd species only n=3!
ILSI Health and
Environmental Sciences
Institute
6

ICH S5 (R2) 2005: Required for Phase III
Fertility & early embryonic development study
Males pre-mating
dosed (2-)4 weeks
1:1 mating & separate M & F when mated.
Continue to dose M until F <strong>are</strong> necropsied
Day 6
stop dosing F
Females dosed 2
wks pre-mating
to implantation
Precoital interval
~Day13 mid-gestation necropsy,
count CL, live & dead implants
Testes weight, histopath, OPTIONAL Fetal morphology – NOT DONE
Sperm analysis – for cause
Toxicokinetics – optional

AZ approach : Male Fertility
Assessed within 3 or 6 month general tox study
Males dosed ~9 weeks
pre-mating, starting
when 5-6* weeks
1:1 mating
Recovery
UNDOSED Females
Remate as needed,
using recovery males if included
Precoital interval
~Day13 “headcount” necropsy,
count CL, live & dead implants
General tox design full dataset: TK, clin path, full tissue list.
Lose 1 week food consumption data. No confusion over affected gender!
* Choose ~ 5 weeks start age to support paeds indications

Undosed females & General Tox test males:
% mated within 7 nights (21 studies, 1684 females)
Number
Of Studies
90 * 95* 97 98 99 100
ILSI Health and
Environmental Sciences
Institute
*CRO with slight smear technique issue!
9

Pregnancy rates in control females mated to
control males in general tox studies
98% chance <strong>of</strong> have > or = 15/16 pregnant in a control group <strong>of</strong> 16
ILSI Health and
Environmental Sciences
Institute
10

Live embryo numbers in control
Wistars mated to General Tox males
Live embryos
ILSI Health and
Environmental Sciences
Institute
11

Power (%) to detect reductions in number <strong>of</strong> live
embryos – using control data simulations
P Jarvis, T Mitchard, J Stewart (in progress)
Confirming adequacy <strong>of</strong> n = 15 /16 to assess male fertility
ILSI Health and
Environmental Sciences
Institute
12

<strong>How</strong> <strong>are</strong> <strong>the</strong> <strong>Possible</strong> <strong>Effects</strong> <strong>of</strong> <strong>Paternal</strong> <strong>Drug</strong>
<strong>Exposure</strong> on Pregnancy Outcome Addressed
during <strong>Drug</strong> Development?
•Pre FTiM package
•Rodent mating study options
•Clinical trials sequelae
•Areas <strong>of</strong> uncertainty
ILSI Health and
Environmental Sciences
Institute

<strong>How</strong> do preclinical data influence clinical trials?
2009 Informal Industry Survey re male contraception
• Informal PhRMA survey (Tony DeLise) – 9 respondent companies
• 5/9 did NOT have written policy, but all react to male reprotox signals
with advice on procreation.
• Is condom use <strong>the</strong> default? 8/9 – NO
2 companies - issue driven eg genetic toxicants or teratogens
• 3/ 9 reported requests for seminal fluid concentrations <strong>of</strong> teratogens
• 6/9 reported requests for use <strong>of</strong> male condoms based ei<strong>the</strong>r on lack
<strong>of</strong> EFD data or evidence <strong>of</strong> teratogenicity
ILSI Health and
Environmental Sciences
Institute
14

Areas <strong>of</strong> regulatory & clinical uncertainty
Male procreation and use <strong>of</strong> condoms
• Confusion: risk to WCBP/ pregnancy from exposure to drug in ejaculate
– In absence <strong>of</strong> EFD data, should men use condom if partner is not using contraception?
– For compounds deemed “high risk” <strong>of</strong> causing fetal harm, is it justified to continue to ask
men to use condom ? What data <strong>are</strong> needed to remove need for condom?
– (If we believe <strong>the</strong>re is risk <strong>of</strong> sufficient absorption to affect fetus, should we be concerned
re. o<strong>the</strong>r undesirable PD affects in sexual partners <strong>of</strong> male patients?)
• Confusion : management <strong>of</strong> potential for male mediated effects
– If genotox negative and no effects on testes path, until mating studies <strong>are</strong> completed and
proven negative, should men be required to avoid procreation ? And for how long?
– What if mating studies <strong>are</strong>n’t feasible in a relevant species?
ILSI Health and
Environmental Sciences
Institute
15

Areas <strong>of</strong> regulatory & clinical uncertainty
Instruction to avoid procreation ≠ condom use
ILSI Health and
Environmental Sciences
Institute
“XXX may affect your fertility.” Or “<strong>the</strong> effect <strong>of</strong> XXX on fertility is unknown”
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Do not attempt to fa<strong>the</strong>r children while taking XXX or for YY months after stoppingTx”.

Areas <strong>of</strong> regulatory & clinical uncertainty
Duration <strong>of</strong> restriction on procreation / condom use
• MHRA (UK) guidance:
• Contraceptive requirements for male subjects with partners <strong>of</strong>
childbearing potential should be included in <strong>the</strong> protocol where needed.
These requirements should also extend to a suitable period after <strong>the</strong> last
dose <strong>of</strong> study medication (e.g., a whole spermatogenic cycle or five halflives)
and should be based upon <strong>the</strong> availability and results <strong>of</strong> reproductive
toxicity data.
• Above is interpreted in different ways.
ILSI Health and
Environmental Sciences
Institute
17

Areas <strong>of</strong> regulatory & clinical uncertainty
Condom use when partner is/may be pregnant
• MHRA (UK) guidance:
• Where <strong>the</strong>re is a risk <strong>of</strong> drug secretion through <strong>the</strong> ejaculate, male
subjects whose partners <strong>are</strong> pregnant should use condoms for <strong>the</strong>
duration <strong>of</strong> <strong>the</strong> study and for a suitable time afterwards (e.g., five halflives).
This is to ensure that <strong>the</strong> fetus is not exposed to <strong>the</strong> IMP through
vaginal absorption.”
• Above (unintentionally?) independent <strong>of</strong> animal EFD study results
• <strong>Drug</strong> likely to be detectable in ejaculate - does amount matter?
ILSI Health and
Environmental Sciences
Institute
18

Areas <strong>of</strong> scientific uncertainty:
Risk <strong>of</strong> embry<strong>of</strong>etal harm from drug exposure in ejaculate
• Absorption from vagina (& caudal rectum) avoids “1 st pass” metabolism.
• Klemmt & Scialli (2005) BDR 74:119-131 “The Transport <strong>of</strong> Chemicals
in Semen” . <strong>Drug</strong> concentration in seminal fluid < x10 blood
• Assume ejaculate conc is x10 blood & 100% bioavailability & daily
sexual intercourse with pregnant partner, predicted concentrations in
female <strong>are</strong> usually

Product information: US versus UK
Bosentan: endo<strong>the</strong>lin antagonist
• US
• Can cause birth defects
• UK
• Can cause birth defects
• Human - reduced sperm counts
• Animal - testes atrophy in long
term studies
• Fertility study OK
• No restriction on male patient
procreation or requirement to
wear condoms
• Male rat fertility study OK
• No restriction on male patient
procreation or requirement to
wear condoms
(No info on semen concentrations in ei<strong>the</strong>r )
ILSI Health and
Environmental Sciences
Institute
20

Future state?
Genotoxic?
No
Testicular
toxicant?
YES
YES
1. Wear condom during trial
and for x5 half life washout period
2. Avoid procreation for (6) months
3. Consider sperm banking
Avoid procreation for (3) months
(Consider sperm banking)
No
Mating study
negative ?
NO or UNKNOWN
Avoid procreation during trial and
for (2) weeks afterwards
21

HESI DART <strong>Drug</strong>s in Human Semen
Team & Acknowledgments
•AZ Male Fertility Data Modelling:
Philip Jarvis and Terri Mitchard
•HESI DART Steering Team
Graham Bailey
Kimberly Benson
Bruce Beyer
Bill Breslin
Gary Chellman
Ruediger Cordts
Liz Davidson
Tony DeLise
Wafa Harrouk
Kok Wah Hew
Julia Hui
Johnson & Johnson
U.S. FDA
san<strong>of</strong>i-aventis
Lilly
Charles River Labs
Boehringer-Ingelheim
Consultant (MHRA)
san<strong>of</strong>i-aventis
U.S. FDA
Takeda
Celgene
Jim Kim
HESI
Larry Leshin U.S. FDA
Jimmy McBlane MHRA
Graeme M<strong>of</strong>fat Amgen
Christine Nguyen U.S. FDA
Anthony Scialli Tetra Tech Sciences
George Scott Amgen
Jane Stewart AstraZeneca
Kary Thompson Bristol-Myers Squibb
Ulla Wandel-Liminga Sweden Medical Products Agency
ILSI Health and
Environmental Sciences
Institute
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