Nintedanib

All animal models were performed twice and data was jointly analyzed.
All animals treated were included in the analysis. Interventions were not
blinded, but analysis of animal samples was. a)
Bleomycin-model:C57Bl/6, 9–12 weeks-old, female mice were
purchased (Taconic Biosciences, Hudson, NY). Knockout mice
(Dio2^−/−),
Ppargc1a^−/− and
Pink1^−/− of C57/BL6 background
were obtained from Jackson Lab (Bar-Harbor, ME). As previously
published^{11 (link),53 (link)} genetic deletion of either PINK1 or
PPARGC1A does not produce any endogenous lung phenotype and is not associated
with derangements of lung physiology.
Ppargc1a^−/− and
Pink1^−/− mice are not
embryonically lethal, they produce litters of appropriate Mendelian size, are
fertile and healthy and present with no gross morphology abnormalities. Mice
were anesthetized by placing them in a chamber having paper towels soaked with
40% isoflurane solution diluted with 1,2-propanediol. Mice were randomly
assigned to either intratracheal 1.5 U/kg of bleomycin (Hospira, IL) or
equivalent volume (50 μL) of 0.9% normal saline was administered
intratracheally as previously described^{54 (link),55 (link)}. To test
therapeutic efficacy of TH in bleomycin-induced established fibrosis we used
intraperitoneal T4 (T2376-Sigma Aldrich, 100μg/kg), or aerosolized T3
(T2877-Sigma Aldrich, 40 μg/kg). T4 was administered systemically at
days 10, 12, 14, 16 and mice were sacrificed on day 19, following
bleomycin-challenge. Aerosolized T3 was administered every other day at days
10–20 and mice were sacrificed on day 21. Dose regimens were based on
previously published protocols^{24 (link),56 (link)}. Equivalent
volumes of normal saline 0.9% were used as controls in all approaches.
For survival analysis we used a double dose of bleomycin (3.0 U/kg) and
administered aerosolized T3 (40 μg/kg) or normal saline 0.9% at
days at days 10–20 (established fibrosis) following challenge and
survival data was collected at day 21, respectively. Mice were randomly assigned
to receive either pirfenidone (100 mg/kg) or nintedanib (60 mg/kg) or vehicle
(0.9% saline) via oral gavage, as previously described⁵⁷ , at days 10–20 (on a
daily basis) following bleomycin administration and mice were sacrificed at day
21. Aerosolized delivery of T3 was performed following a standardized protocol.
Briefly: T3 was diluted to a final concentration of 40 μg/kg in 6 ml of
PBS and suspension was aerosolized using a conventional aerosol nebulizer
(Omron) throughout a chamber that allowed simultaneous exposure of 8 mice for 30
minutes until mist stopped forming in the nebulizer chamber. b)
TGF-β1-induced lung fibrosis: Inducible lung targeted
TGF-β1-overexpressing triple transgenic mice
(CC10-rtTA-tTS-TGF-β₁₎ generated as previously
described^{16 (link)} were used.
Briefly: A triple transgenic system based on the tetracycline-controlled
transcriptional suppressor (tTS) and the reverse tetracycline transactivator
(rtTA) was constructed. In this system, the CC10 promoter constitutively drives
the expression of rtTA and tTS in a lung-specific fashion. In the absence of
dox, tTS binds to and actively suppresses the expression of the
tet-O–regulated TGF-β1 transgene. In the presence of dox, tTS is
released allowing the activating, dox binding rtTA to bind to the tet-O and
activate transgene expression. As expected induced TGF-β₁– overexpression upon addition of doxycycline water on a daily basis
(days 0–20) caused airway and parenchymal fibrotic response as assessed
by increased lung collagen deposition indicated by hydroxyproline levels and
Masson Trichrome staining. Aerosolized T3 (40 μg/kg) or normal saline
0.9% was administered every other day on days 10–20 following
addition of doxycycline and mice where sacrificed on day 21. c) Sobiterome
therapeutic protocol: Sobiterome was provided by the laboratory of
Dr. Scanlan as previously described^{21 (link),58 (link)}. C57Bl/6,
9–12 weeks-old, female mice were randomly assigned to be challenged with
1.5 U/kg of bleomycin or equivalent volume of normal saline 0.9% at day
0. Mice were then randomly assigned to treatment with 5 mg/kg of sobiterome
diluted in 50 μL of normal saline 0.9% or vehicle (equivalent
volume of normal saline 0.9%) administered by oral gavage at days 10,
12, 14, 16 and 18 following bleomycin administration and mice were sacrificed on
day 21.