Supplemental document
Table S1: In vitro permeability measurements through porcine buccal and sublingual
tissues for 9 model compounds
Model compounds
Drug name
Molecular
(g/mol)
Zolmitriptan
287.36
weight
pKa
logDpH=7.4
logP
0.868
2.4
Base:8.94;
Acid:10.97, 15.64
Atenolol
266.34
Base: 9.34
-1.015
0.56
Metoprolol
267.37
Base: 9.44
0.047
1.96
Asenapine
285.8
Base: 7.93
3.988
4.63
Propranolol
259.35
Base:9.3
1.177
2.96
Verapamil
454.61
Base: 8.1
3.851
4.63
Zolpidem
307.4
Base: 4.28
3.06
3.06
MK-compound A
420.5
Base <1.8
2.78 (Pka 1.8)
2.78
Amitryptiline
277.41
Base: 9.13
3.244
4.98
All properties are predictions from ADMET Predictor v6.0
Tissue preparation
The porcine sublingual and buccal tissues were obtained from animals sacrificed at a local
slaughterhouse (Lansdale, PA) and were transported to the laboratory within 1 hour. The
majority of underlying muscles and connective tissues were surgically removed from sublingual
and buccal tissues with scissors before storage at -70 °C. The tissues were dry-wrapped in double
aluminum foil to avoid dehydrating during storage. Tissues were thawed in incubator at 37 °C
for 30 min prior to the permeation study on the date of the experiment. Any underlying muscles
and connective tissues were carefully removed until only epithelium remained.
Tissue thickness and integrity
Tissue thickness and electrical resistance were measured as quality control for tissue trimming
and integrity. The peripheral parts of sublingual and buccal tissues which were mounted on
Ussing chambers were measured by caliper. Electrical resistance was measured – ideal values
should be within 100-1000 Ω/cm2.
Permeation studies
In vitro transport was studied using the Multichannel Ussing Chambers System (Navicyte,
Warner Instruments) at 37 °C. 3H or 14C radiolabelled compound were used in donor chamber.
The Ussing cells have the diffusion area=1.78 cm2. Sublingual and buccal mucosal were thawed
in PBS solution for 10 minutes at room temperature. Each tissue was spread over an Ussing
chamber and clamped with the epithelial side facing the donor compartment. Donor and receiver
chambers were filled with 5 ml of drug solution and PBS buffer, respectively (pH 7.4).
Compound donor solutions were prepared in PBS buffer and the concentration in the donor
chamber was 3 micro curie (the real mole concentration in donor solution depends on the
concentration of radiolabelled stock solution). Oxygen was bubbled through the diffusion system
at both donor and receiver sides to minimize the aqueous diffusion layers and facilitate mixing.
Diffusion experiments (n≥6) were conducted for 7 or 8 h. Samples (500 µl) were collected from
the receiver chamber and replaced with the same volume of fresh PBS buffer. 20 µl of donor side
solution was collected at 0 and 6 h.
Sample Analysis
500 µl samples collected from receiver chamber (20 µl from donor side) were mixed with 5 ml
biodegradable counting cocktail and vortexed for 15 seconds. The mixture was analyzed by
liquid scintillation counting.
The cumulative amount (Q, μmol) of model compounds permeated across sublingual/buccal
epithelium was calculated by the following equation.
Q = C𝑛 × V + 0.5 × ∑𝑛−1
𝑖=1 C𝑖
(1)
Where V is the volume of the receptor cell, Cn is the drug concentration at time point ‘n’, and Ci
is the model compound concentration at time point ‘i’. The radioactivity was converted to
molarity based on the conversion factor.
The cumulative amount (μmol) of compound permeated across sublingual/buccal epithelium was
plotted versus time (h). Each data presents as mean ± SD of six determinations.
Figure S1: Permeation results of 9 model compounds through porcine buccal and
sublingual tissues
Zolmitriptan in buccal tissue
Accumulated Amounts (µmol)
2.5E-03
2.0E-03
1.5E-03
1.0E-03
5.0E-04
0.0E+00
0
4
Time (hr)
6
8
Zolmitriptan in sublingual tissue
5.0E-02
Accumulated Amounts (µmol)
2
4.0E-02
3.0E-02
2.0E-02
1.0E-02
0.0E+00
0
2
4
Time (hr)
6
8
Atenolol in buccal tissue
Accumulated Amounts (µmol)
1.2E-07
1.0E-07
8.0E-08
6.0E-08
4.0E-08
2.0E-08
0.0E+00
0
2
4
6
8
Time (hr)
Atenolol in sublingual tissue
Accumulated Amounts (µmol)
1.2E-07
8.0E-08
4.0E-08
0.0E+00
0
2
4
Time (hr)
6
8
Metoprolol in buccal tissue
Accumulated Amounts (µmol)
1.0E-07
8.0E-08
6.0E-08
4.0E-08
2.0E-08
0.0E+00
0
2
4
6
8
Time (hr)
Amitryptiline in sublingual tissue
Accumulated Amounts (µmol)
1.0E-06
8.0E-07
6.0E-07
4.0E-07
2.0E-07
0.0E+00
0
2
4
Time (hr)
6
8
MK compound A in buccal tissue
Accumulated Amounts (µmol)
1.0E-06
8.0E-07
6.0E-07
4.0E-07
2.0E-07
0.0E+00
0
2
4
6
8
Time (hr)
MK compound A in buccal tissue
Accumulated Amounts (µmol)
3.0E-06
2.5E-06
2.0E-06
1.5E-06
1.0E-06
5.0E-07
0.0E+00
0
2
4
Time (hr)
6
8
Propranolol in buccal tissue
Accumulated Amounts (µmol)
1.0E-06
8.0E-07
6.0E-07
4.0E-07
2.0E-07
0.0E+00
0
2
4
6
8
Time (hr)
Propranolol in sublingual tissue
Accumulated Amounts (µmol)
1.5E-06
1.2E-06
9.0E-07
6.0E-07
3.0E-07
0.0E+00
0
2
4
Time (hr)
6
8
Asenapine in buccal tissue
Accumulated Amounts (µmol)
4.0E-04
3.0E-04
2.0E-04
1.0E-04
0.0E+00
0
2
4
6
8
Time (hr)
Asenapine in sublingual tissue
Accumulated Amounts (µmol)
8.0E-04
6.0E-04
4.0E-04
2.0E-04
0.0E+00
0
2
4
Time (hr)
6
8
Verapamil in buccal tissue
Accumulated Amounts (µmol)
8.0E-08
6.0E-08
4.0E-08
2.0E-08
0.0E+00
0
2
4
6
8
Time (hr)
Verapamil in sublingual tissue
Accumulated Amounts (µmol)
3.6E-07
3.0E-07
2.4E-07
1.8E-07
1.2E-07
6.0E-08
0.0E+00
0
2
4
Time (hr)
6
8
Zolpidem in buccal tissue
Accumulated Amounts (µmol)
2.0E+00
1.6E+00
1.2E+00
8.0E-01
4.0E-01
0.0E+00
0
2
4
6
8
Time (hr)
Table S2: Key model input parameters for prediction of Fa_IO in asenapine, verapamil,
propranolol, and nicotine. Values of fut and diffusivity were calculated using Equation 6
and 7
Dose
Compounds
(mg)
Dosage
forms
logDpH=7.4
pKa
(base)
Reference
Solubility
(mg/ml)
fut
Diffusivity
(cm2/s)
Asenapine
5
Sublingual
Tablet
3.99
7.93
3.7
pH = 4.6
0.059
1.12 × 10-6
Verapamil
40
Sublingual
Solution
3.85
8.10
0.0162
(pH = 8.8)
0.063
1.12 × 10-6
Propranolol
40
Sublingual
Tablet
1.18
9.30
1.66
(pH=10.5)
0.26
5.54 × 10-7
Nicotine
2
Sublingual
Tablet
-2.48
8.51
125
(pH=11.2)
0.52
1.52 × 10-7