healthmedicinet

Culturing of primary human keratinocytes

Fresh human abdominal skin, obtained from a Caucasian donor undergoing an abdominoplasty
(female aged 46 years old) was collected and maintained in a holding medium consisting
of HEPES buffered DMEM containing antibiotics and antifungals. The epidermis was removed
from the dermis after overnight incubation at 4 °C with dispase in the holding medium
and then underwent trypsin digestion. The keratinocyte suspension was seeded on 3 T3
fibroblasts and rendered mitotically inactive by mitomycin C treatment. Human keratinocyte
monolayers were cultured for 6 days in DMEM (2 mM Ca
²⁺
), with 10 % FCS, L-glutamine (2 mM), insulin (5 ?g/mL), hydrocortisone (0.4 ?g/mL),
epidermal growth factor (EGF, 10 ng/mL), penicillin (100 IU/mL), and streptomycin
(100 ?g/mL), at 37 °C, in 95 % air/5 % CO
₂
atmosphere, with 95 % relative humidity. After 6 days, the 3T3 feeder cells were removed
from the human keratinocyte cultures by a trypsin/EDTA treatment. The next day, the
human keratinocytes were removed by trypsinization. The human keratinocyte suspension
was centrifuged, resuspended in fresh culture medium, and then strained through a
series of filters. Cells (1.5×10
⁵
/well) were then seeded into 96-well plates with 200 ?L of EpiLife® medium and 60 ?M
calcium, antibiotics, and human keratinocyte growth supplement (HKGS) per well. Human
tissue samples for the isolation of keratinocytes (described above) and skin explants
(described below) were obtained in accordance with the Human Tissue Act of 2004 (England
Wales and Northern Ireland) and collected with the appropriate donor consent.

Treatment and harvesting of primary human keratinocytes

RTA 408 (3, 30, 100, 300, 700, and 1000 nM), vehicle (DMSO, 0.1 % final concentration),
or nothing (media control) was incubated with cells in two separate 96-well plates
(1.5 x 10
⁵
cells per well) for 16 h. At the time of harvest, one plate utilized the MTT assay
(Invitrogen, V13154) to examine cell viability, and one plate was processed for QuantiGene
Plex 2.0 analysis (i.e., mRNA expression of Nrf2 target genes), according to manufacturer’s instructions
and as previously reported 9], 11].

Culturing of human skin explants

Skin, obtained from a female reduction mammoplasty (48 year old donor), was collected
and placed in a holding medium consisting of HEPES buffered DMEM containing antibiotics
and antifungals. The fat was removed, and a 5-mm punch biopsy was used to cut 30 biopsies.
Biopsies were cultured at the air-liquid interface with 5 mL of DMEM media (2 mM Ca
²⁺
), 10 % FCS, L-glutamine (2 mM), insulin (5 ?g/mL), hydrocortisone (0.4 ?g/mL), EGF
(10 ng/mL), penicillin (100 IU/mL), and streptomycin (100 ?g/mL), at 37 °C, in a 95 %
air/5 % CO
₂
atmosphere, with 95 % relative humidity. The dermis of each culture was immersed in
the media, while the epidermis was in contact with air.

Ex vivo treatment and harvesting of human skin explants

Skin cultures were split into 5 treatment groups. RTA 408 (0.03, 0.3, or 3 %), vehicle
(sesame oil), or nothing (media control) was applied topically twice daily for 2 days
and once on Day 3. Approximately 50 ?L of RTA 408 or vehicle was applied to the entire
surface of the skin cultures. Prior to each application, a visual inspection of the
skin cultures confirmed there was no residual RTA 408 or vehicle from the previous
administration. All skin cultures were harvested 8 h after the final administration
on Day 3, with half of the replicates fixed for 24 h in phosphate-buffered formalin
(pH 7.0–7.4), transferred to 70 % ethanol, then processed and paraffin-embedded, according
to standard histological techniques. The remaining skin samples were snapped frozen.

Quantigene 2.0 Plex mRNA expression analysis

Messenger RNA (mRNA) was quantified using Quantigene Plex 2.0 technology according
to manufacturer’s protocol (Affymetrix, Inc., Santa Clara, CA) and as previously described
12]. Probe sets were designed against the human genome for analysis of Nrf2 target genes,
and a modified version of Panel 11834 (Affymetrix) was used. Human primary keratinocyte
data were normalized to the housekeeping gene PPIB. Human skin explant data were normalized
to the average of housekeeping genes RPL13A and PPIB.

Immunohistochemical analysis of NQO1 protein in cultured human skin explants and biopsies

Levels of NQO1 protein in skin sections were determined by immunohistochemistry (IHC)
using previously described methods 9]. NQO1 staining intensity of 5X magnification photomicrographs was quantified using
ImageJ software v1.46 with the Densitometry 1 plug-in, both freely available from
the National Institutes of Health (http://rsbweb.nih.gov/ij/index.html).

Healthy volunteer clinical study design

The clinical study (https://clinicaltrials.gov/ct2/show/NCT02029716) enrolled healthy adults (male and female) aged 18 to 65 years, with Fitzpatrick
skin type I to IV, and a body mass index (BMI) between 18 and 32 kg/m
²
. Demographic data are presented in Table 1. The study was conducted sequentially in 3 parts to assess the safety, tolerability,
PD, and PK of RTA 408 Lotion applied topically twice daily (BID), at 8:00 a.m. and
8:00 p.m., for up to 28 days. For each application, the appropriate amount of lotion
was applied to the skin and gently massaged for the appropriate time (Parts A and
B: 10–15 s; Part C: 45 s). The areas of application were allowed to dry for 5 min,
and then the entire area was covered with loose-fitting gauze to keep the lotion confined
during normal activities.

Table 1. Phase 1 Clinical trial healthy volunteer baseline characteristics

Part A was a randomized, double-blind, placebo-controlled assessment of the safety,
local skin tolerability, PD, and PK of 3 concentrations of RTA 408 Lotion (0.5 %,
1 %, and 3 %, w/w) compared to lotion vehicle (0 %) applied topically to 12 healthy
subjects BID for 14 days to a small skin surface area on the lower back (4 cm
²
for each concentration). Part B was open-label and assessed the safety, tolerability,
PD, and PK of the highest tolerated dose of RTA 408 Lotion from Part A (i.e., 3 %) applied topically to 10 healthy subjects BID for 14 days to a larger skin surface
area on the lower back (100 cm
²
). During the dosing period in Parts A and B, subjects were confined to the study
site for 15 days. Part C was open-label and assessed the safety, tolerability, PD,
and PK of RTA 408 Lotion (3 %) concentration applied BID to an even larger skin surface
area (500 cm
²
) on the backs of 10 healthy subjects for 28 days. During the dosing period in Part
C, subjects were confined to the study site for 29 days. Total daily doses of RTA
408 for Parts A, B, and C were approximately 1.8, 30, and 150 mg/day, respectively.
The assessment of safety was based primarily on the incidence, intensity, and type
of adverse events, Modified Draize Skin Irritation Assessments, clinical laboratory
assessments (hematology, clinical chemistry, and urinalysis), physical examinations,
12-lead electrocardiograms (ECG), and vital signs.

Analysis of RTA 408 plasma concentrations in healthy volunteers following topical
administration of RTA 408 lotion

Blood samples for PK analysis were collected for determination of plasma RTA 408 concentrations
prior to dosing and 1, 2, 4, 12, and 24 h after the first topical application of RTA
408 Lotion on Days 1, 7, and 14 of Parts A, B, and C and also on Day 28 for Part C.
A validated LC/MS/MS method with a lower limit of quantitation (LLOQ) of 0.074 ng/mL
and an upper limit of quantitation (ULOQ) of 37.0 ng/mL was used for quantification
of RTA 408 in plasma samples.

Analysis of NQO1 protein expression in healthy volunteer skin punch biopsies

Punch biopsies (3 mm) for evaluation of induction of NQO1 protein expression, the
prototypical Nrf2 target gene, were collected the day following the final dose in
each part of the study. A local injection of lidocaine HCl (1 %) was used for anesthesia.
Biopsies were incubated in formalin at room temperature for 24 h and then transferred
to 70 % ethanol. NQO1 IHC on the skin biopsies was performed as described above.

The protocol and informed consent documents were submitted to and approved by the
duly constituted Western Institutional Review Board prior to initiation of the clinical
study. The study was conducted in accordance with the Declaration of Helsinki and
with all applicable laws and regulations of the locale and country where the study
was conducted, and in compliance with Good Clinical Practice Guidelines.

Statistics

Nrf2 target gene data were analyzed with Sigmaplot 12.0 (Systat, Inc., San Jose, CA)
by student’s t-test or by one way-analysis of variance (ANOVA) followed by Duncan’s Multiple Range
post-hoc test with significance set at p??0.05.