Hydroxyethylcellulose

This study conformed to the National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. It was approved by the Experimental Animal Administration Committee of Tianjin Medical University and Tianjin Municipal Commission for Experimental Animal Control.
HFD and low-dose STZ treatment were used to induce type II diabetes mellitus in rats [8 (link)]. The HFD + STZ model, which similar demonstrates a progression from insulin resistance to hypoinsulinaemia and hyperglycemia, mimics the natural T2DM pathogenesis in humans and is suitable to investigate the pathogenesis of diabetic complications and test the efficiency of anti-diabetic agents. A total of 96 adult male Sprague–Dawley (SD) rats (200 ± 20 g) were purchased from the HuaFuKang Bioscience Co., LTD (Beijing, China). They were kept under a 12 h light/dark cycle at room temperature (20–22 °C) and humidity (50–60%). After 1 week, the rats were divided into two groups: HFD group (n = 72) and control group (n = 24). The rats of the HFD group were fed high-fat chow (H10060, fat energy ratio = 60 kcal%, protein energy ratio = 20 kcal%, carbohydrate energy ratio = 20 kcal%, the Beijing HuaFuKang Bioscience Co., LTD, China) for 4 weeks, then given a single tail vein injection of STZ (30 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) dissolved in citrate buffer at pH 4.5. The rats of control group were fed regular chow and were injected with the same dose of citrate buffer. One week following the STZ injection, blood samples were collected from the tail vein to measure the blood glucose level. The blood glucose level of control group rats was kept within the normal range. HFD group rat with random blood glucose > 16.7 mmol/L was considered successful induction of DM, and was used for further investigation [9 (link)]. The same dose of STZ was injected again in rats with blood glucose level that did not meet the diagnostic criteria. The remaining rats that failed to meet the diagnostic criteria after the injection were excluded from the study. This process was repeated until a sufficient number of DM animals were produced. Blood glucose concentration of the DM models was monitored weekly using the glucometer Optium Xceed (Abbott Laboratories MediSense Products).
The rats were then divided into four groups: control group (CON, n = 24); DM group (DM, 0.5% hydroxyethylcellulose/day, intragastric administration, ig, n = 24); low dose of EMPA (low-EMPA, 10 mg/kg/day, ig, n = 24); and high dose of EMPA (high-EMPA, 30 mg/kg/day, ig, n = 24). The dose of empagliflozin was based on the previous studies [10 (link), 11 (link)]. Empagliflozin was supplied by Boehringer Ingelheim Pharma GmbH & Co. (KG, Germany). Besides from the control group, the remaining three groups were composed of the DM models. The rats were treated for 8 weeks. All rats were anesthetized with sodium pentobarbital by intraperitoneal injection and sacrificed following weeks of treatment. The first 8 rats of each group were used for the first part of the experiments (including echocardiographic, hemodynamic, histological, and serum biochemical and oxidative stress-related markers examination, and western blot analysis). The next eight rats were used for the electrophysiological studies, and the remaining eight rats were used for examinations of mitochondrial function.

Culture medium was quickly aspirated from cells grown on 6 cm culture dishes, and cells were washed with 1 ml of ice-cold PBS. After rapid aspiration of PBS, a minimal volume of ice-cold 5% perchloric acid was added and the samples vortexed to ensure complete lysis. After centrifugation (14,000 rpm, 4°C, 3 min) to remove acid-insoluble material, the supernatant was extracted with two washes of an equal volume of 1:1 tri-n-octylamine and 1,1,2-trichlorotrifluoroethane. The nucleotides remaining in the aqueous phase were then separated by capillary electrophoresis with on-column isotachophoretic concentration, using run buffers consisting of 50 mM Na phosphate and 50 mM NaCl (pH 5.2; leading buffer) and 100 mM MES/Tris (pH 5.2; tailing buffer). To each buffer was added 0.2% hydroxyethylcellulose to decrease electro-osmotic flow. Nucleotide peaks were detected by UV absorbance at 260 nM and integrated using System Gold software (Beckman). Nucleotide ratios were calculated from peak areas after correction for retention times. Identification of peaks as ATP, ADP, and AMP were confirmed by additional runs spiked with internal standards and analysis of absorbance spectra of individual peaks.

OCT was performed using a Micron^® IV (Phoenix Research Labs, Pleasanton, CA) with a contact lens specifically designed for rat OCT. In the RCS^-/- rats, OCT was conducted at 12 time points between postnatal (PN) day 17 and PN day 111, while in the RCS^+/+ rats, OCT was carried out at eight time points between PN day 18 and PN day 67. At each time point, four to eight rats (eight to 16 eyes) were evaluated. The rats were anesthetized by intraperitoneal injection of a mixture of medetomidine hydrochloride (0.315mg/kg), midazolam (2.0mg/kg), and butorphanol tartrate (2.5mg/kg). To alleviate the pain associated with injection, the rats were pre-anesthetized by inhalation of 80% carbon dioxide and 20% oxygen prior to the intraperitoneal injection. The researchers monitored the physical conditions of the rats including heart beat and respiratory pattern, by inspection and gentle palpation every minute during the experiment. The pupils were dilated using eyedrops that contained a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride. The corneal surface was protected using a 1.5% hydroxyethylcellulose solution. The rat ocular fundus was monitored using the fundus camera of the Micron^® IV, and the position of the retinal OCT image was set horizontally at one disc diameter superior to the optic disc. Fifty images were averaged to eliminate projection artifacts. The acquired OCT images were quantitatively analyzed using the InSight^® software (Phoenix Research Labs). Five images from five rats in each genotype group were selected at each time point on the basis of image sharpness; importantly to avoid selection bias, the pictures were not selected by thickness or reflectivity. We measured the thicknesses of the inner (A, Fig 1), middle (B, Fig 1), and outer (C, Fig 1) layers of the neural retina, as well as that of the combined RPE and choroid (D, Fig 1). The middle layer consists of the combined outer plexiform and outer nuclear layers, and the outer layer consists of the photoreceptor inner segment (IS) and OS layers (Fig 1).

Since DFO is hydrophilic, the development of the cream formulation involved incorporating Transcutol P (Gattefosse) to create emulsions which allow for effective penetration of the stratum corneum layer of the skin. Transcutol P is a highly purified form of diethylene glycol monoethyl ether (DEGEE) that serves as a safe solubilizer and enhancer for transdermal drug delivery.
²² (link) Transcutol P increases drug solubility in the vehicle (water) and penetration through the stratum corneum without disrupting physiologic lipid bilayer skin structures. Transcutol P has been approved by FDA as an inactive ingredient and has already been used in a variety of FDA approved human and veterinary drug products.
²² (link) Hydroxyethylcellulose was used as a gelling agent and mixed with water before finally being mixed with Transcutol P and DFO overnight in an overhead mixer to create the emulsion. The DFO cream was found to release approximately 46% at 6 h and approximately 83% after 24 h.

In comparison to the referenced Cystadrops^®, a compounded eye drop formulation containing the same amount of CYA was prepared from the available compounds that are officially on the positive list of the National Institute of Pharmacy and Nutrition, Hungary (Table 1). Along with being on the positive list, hydroxyethyl cellulose (viscosity: 250–400 mPa·s, Ph.Hg. VIII) acts as a demulcent by relieving eye inflammation, irritation, and dryness [23 (link)]. The applied polymer concentration enabled sterile filtration. The preparation of the solution was conducted in a laminar air flow cabinet under aseptic conditions consisting of four main steps: (1) preparation of the buffer solution, (2) mixing of hydroxyethylcellulose into a portion of the buffer solution to produce a viscous solution, (3) dissolution of the active substance and other excipients in the remaining buffer solution, and (4) mixing of the two solutions followed by sterile filtration through a sterile polyethersulfone (PES) filter membrane of 0.22 µm pore diameter and 25 mm diameter (Stericup^®, Merck-Millipore, Darmstadt, Germany).