Sulfated caffeic acid dehydropolymer attenuates elastase and cigarette smoke extract-induced emphysema in rats: sustained activity and a need of pulmonary delivery
Bhawana Saluja1, Hua Li1, Umesh R. Desai2, Norbert F. Voelkel3 and Masahiro Sakagami1§
1Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University, 410 N. 12th Street, Richmond, Virginia 23298, USA
2Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, 800 East Leigh Street, Richmond, Virginia 23219, USA
3Pulmonary and Critical Care Medicine Division and Victoria Johnson Center for Obstructive Lung Diseases, Virginia Commonwealth University, 1220 East Broad Street, Richmond, Virginia 23298, USA
§Corresponding author:
Masahiro Sakagami, Ph.D.
Department of Pharmaceutics
School of Pharmacy
Virginia Commonwealth University
410 N 12th Street, P.O. Box 980533
Richmond, VA 23298-0533, USA
Phone: +1-(804)828-2014
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Supplemental Information
S-1 Orotracheal instillation and subcutaneous injection
Orotracheal (OT) instillation was performed under isoflurane anesthesia to dose saline, HSE/CSE or CDSO3 to the lung. At each dosing, rats were exposed to 4 % isoflurane (Webster Veterinary Supply, Sterling, MA) vapor for 4 min, generated from the Ohmeda Tech 4 Surgivet® vaporizer (Smiths Medical North America, Waukesha, WI) in 95 % oxygen and 5 % air of the carrier gas at 3.5 l/min. The animals were then placed on a board inclined at 60 degrees, and 0.2 or 0.1 ml of HSE/CSE or CDSO3 in saline or saline alone was immediately dosed to the lung as a coarse solution spray using a PennCentury Microsprayer (Wyndmoor, PA) through the oral cavity. The tip of the Microsprayer was inserted through the tracheal lumen to project just before the tracheal bifurcation to ensure consistent delivery and lung-regional distribution. A group of animals (Group 8 in Table 1) received CDSO3 at 30 mg/kg in 0.1 ml via subcutaneous injection to the neck skin fold under the isoflurane anesthesia. Each of the dosing procedures took less than 30 s, after which rats were fully recovered from the anesthesia in 5 min.
S-2 Treadmill exercise endurance
For the lung functional assessments of treadmill exercise endurance, rats were trained from day 1 of the HSE/CSE instillation to run on the AccuPacer Treadmill (AccuScan Instruments, Columbus OH). They were first introduced to learn running against the running belt and recognize electrical foot shock from the bar grid through repeated exposure to the treadmill at a speed of ≤ 5 m/min for ≤ 5 min every day in the first week. From the second week, rats were trained to run against a speed of 10 m/min for 5 min without an inclination every other day. On day 21 and 22 post-HSE/CSE instillation, rats were tested for the exercise endurance on the treadmill to run against a speed of 10 m/min and an inclination of 5 degrees. Exhaustion was judged, when animals received a 5th electrical foot shock from the bar grid or displayed an inability to return the running belt. The exercise endurance was reported as an averaged running time for exhaustion tested on day 21 and 22.
S-3 Airspace enlargement
For the assessments of morphological airspace enlargement, on day 28 post-HSE/CSE instillation, rats completing treadmill studies were killed by exsanguination under surgical anesthesia with an intraperitoneal injection of sodium pentobarbital (50 mg/kg; Nembutal®, Ovation Pharmaceuticals, Deerfield, IL). The left lung lobes were then inflated with ~8 ml of a 45 oC agarose solution (0.5 %; Sigma-Aldrich) introduced through the tracheal cannula at a hydrostatic pressure of 20 cm; the bronchi to the right lung lobes were closed with a suture. After placed on ice for 10 min, the fully inflated left lung lobes were fixed in 10 % buffered formalin (Thermo Scientific) at 4 oC for 24 h. Upon their paraffin block preparation, 4 µm-thick alveolar section slides were prepared and then stained with hematoxylin-eosin. The airspace enlargement was quantified with the mean linear intercept (MLI) values determined by the method originally outlined by Thurlbeck [16] with in-house modifications. In each alveolar section, 5 microscopic field images (3450 x 2585 μm) were randomly selected and printed out. In each image, 5 horizontal lines (3450 μm) were drawn with equal (431 μm) vertical intervals, and their intercepts with the alveolar walls in the line length 3450 μm were counted to determine the MLI values (3450 mm / the number of the intercepts). The MLI value for each animal was thus calculated as an average of the 25 MLI values (5 horizontal lines per image x 5 images).
S-4 A lack of the 2 h preventive effects of caffeic acid (CA)
Caffeic acid (CA; Sigma-Aldrich) is a monomer unit of CDSO3. In our previous in vitro study [10], CA was shown to yield a ~5-fold less potent anti-oxidative activity than CDSO3, while lacking anti-elastase and anti-inflammatory activities. To assess the importance of the triple-inhibitory activities in vivo, CA was tested in the rat model of HSE/CSE-induced emphysema with pulmonary administration at 2 h before the HSE/CSE instillation, as tested with CDSO3. Table S-1 shows the 2 h pretreatment effects of CA at 16 μg/kg, a 10-times greater molar dose of CDSO3 at 30 μg/kg. While the lung tissue rGSH decrease (i.e., oxidative stress) was completely inhibited (98.5 %; p<0.05), neither the anti-elastase nor anti-inflammatory activities was seen, consistent with its in vitro activities [10]. Accordingly, the BALF leukocytes, exercise endurance and MLI values all remained induced, comparable to those in the saline-dosed emphysema (Positive) control animals. This suggested that when emphysema was induced with the mechanisms more than just oxidation, e.g., elastolysis and/or inflammation, like this HSE/CSE-induced model, anti-oxidation alone appeared to be ineffective in preventing the development of emphysema.