Montelukast

Daily diary cards were used from birth to monitor significant troublesome lung symptoms as previously analysed in detail 12 including components of cough, wheeze, and dyspnoea, and use of β2‐agonists, inhaled corticosteroids, and montelukast. Skin symptoms were monitored as active eczema and use of topical steroids. In addition, the diary cards monitored infections, categorized into common cold, pneumonia, pharyngitis, otitis, fever, gastrointestinal infection, and absence from day care institution because of illness (Figure S1). The diary cards were reviewed with the family by the research MD at each visit to validate symptom definitions. All information were subsequently entered into the online database and double‐checked.
Physical examination was performed by the research MD at all scheduled and acute visits, including lung and heart stethoscopy and examination of skin 13, ear, nose, and throat.
CRP measurement was performed at acute visits or when needed using QuickRead 101 (QuickRead Instrument, Orion Diagnostica, Espoo, Finland).
Tympanometric evaluation of the middle ear pressure was performed at yearly visits or when needed (MT10, Interacoustics, Denmark) on both ears.
Spontaneous physical activity was assessed by age 2 years using an omnidirectional accelerometer worn on the ankle for 2 weeks as previously described 16.
Blood pressure was assessed yearly from age 3 years (Welch Allyn Connex: ProBP 3400).
Lung function by multiple‐breath washout (EcoMedics: Exhalyzer D) was assessed from the age of 3 years.
Airway resistance was measured from 3 years of age by whole‐body plethysmography (Master Screen Body; Erich Jaeger GmbH; Würzburg, Germany) 18.
Airflow was measured by spirometry (Vitalograph: Spirotrac II) before and after inhalation of a standard dose of β2‐agonist from age 5 years.
Fractional nitric oxide (FeNO) was measured using an Aerocrine NO system (CLG77AM chemiluminescence analyzer from Ecophysics AG, Duernten, Switzerland) and assessed from age 5 years.

An evidence-based implant-based augmentation mammaplasty was performed, as previously described.^{19 (link)–21 (link)}Periareolar, inframammary fold, and axillary incisions were made under general anesthesia and intravenous sedation for the purposes of preventing visible scarring. Selection of surgical incision is based on our desired outcomes, types of breast implants, the degree of augmentation, the anatomical characteristics of patients, and patient–surgeon preference. Based on the Ranquist formula, we determined the distance extending from the nipple to the inframammary fold, the size of breast implant, and the scope of dissection. After the dissection, each breast was irrigated using a 100 mL of normal saline mixed with H₂O₂ solution at a ratio of 1:1, followed by the use of betadine 100 cc. Then, a breast implant was immersed in a normal saline mixed with ceftezole 1 vial and gentamicin 1 ample and then inserted in a pocket either under the pectoralis major muscle (a submuscular placement) or in the retromammary space above it (a subglandular/submammary placement). Methods for inserting and positioning a breast implant in the pocket were dependent on its types, the degree of augmentation, characteristics of a patient’s body, and our recommendations. Thus, we performed a dual-plane I/II augmentation on a case-by-case basis. Intraoperatively, the patients were intravenously given ceftezole 1.0 g. Incisions were closed using layered sutures in the breast tissue. In addition, skin adhesive or surgical tape were used to close the skin.
Postoperatively, the patients were given cefaclor, nonsteroidal anti-inflammatory drugs, and antacid 3 times daily for a week. Moreover, they were also recommended to take montelukast sodium 10 mg (Lucast tab.; Wooridul Pharmaceutical Ltd., Seoul, Korea) for a month for the prevention of CC and to wear a compressive garment for 3 months. Furthermore, they were also recommended to use an upper or lower band, if necessary, and most of them used an upper one for 1–2 months.
Postoperative course was meticulously monitored during a regular follow-up at 1, 2, 3, and 4 weeks; 3, 6, 9, and 12 months; and thereafter. Moreover, the patients were also recommended to be further evaluated on magnetic resonance imaging scans at 3 years and at a 2-year interval thereafter in accordance with the US Food and Drug Administration labeling recommendation.^{22 (link)}

Patients were randomized to receive antihistamine drug (5 mg oral desloratadine) or a combined therapy with 5 mg oral desloratadine and 10 mg cysteinyl leukotriene receptor antagonist, montelukast. Randomization was stratified according to disease severity (IGA 3 versus 4) [31 (link)] and affected skin region.
The 5 mg of desloratadine was administered to patients in group I. Patients in group II received a combined therapy with 5 mg of the antihistamine agent and 10 mg of montelukast to match the loading AH dose in Group I. o maintain blinding, coded kits containing desloratadine or combined agents were used to mask treatment assignment. Concomitant topical therapy of AD included application of a topical corticosteroid Elocon (Organon Pharma, UK) twice daily, with 12 h’ interval (morning and evening applications). In both groups, the topical corticosteroid was used with 1-month duration. Twice daily, in the period between the steroid applications a special moisturizer, Topicrem DA (La Roche) was used topically. Additionally, patients of both groups were administered thrice per day with a probiotic, Lacto G (GM Pharmaceuticals) for 4 weeks. The clinical parameters were evaluated in all groups before and after the treatment using different therapy outcomes. The treatment outcomes were assessed after a 12-week therapy period.

The permission of the institutional animal ethics committee was obtained before the initiation of the study (Reference approval number- AEC/15/2017). The animals bred in the Central Animal House of the institute [registered under Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA) which is a statutory Committee under the Government Ministry] were used and the study was conducted according to CPCSEA guidelines.
Experimental animals: The study was carried out in Wistar rats of either sex, each weighing between 150 and 250 grams. The rats were housed in polypropylene cages with stainless steel top grills having facilities for providing food and water. The rats had free access to UV-filtered water and food which was administered in the form of pellets. Paddy husk was used as the bedding in the cages. Regulated conditions were maintained with temperature 23 ± 4°C, humidity 30%–70% and 12-hour light-dark cycles.
Study drugs and chemicals: Disease-inducing chemical agent Streptozotocin (STZ) (Product number-572201), study drugs Montelukast (Product number-PHR-1603-1G) and Enalapril (Product number-PHR-1603-1G) were purchased from Sigma-Aldrich. STZ was administered intraperitoneally at a dose of 60 mg/kg in 0.1 M sodium citrate buffer (pH-4.5). Montelukast and Enalapril were given orally mixed in normal saline (0.9%) and 0.5% carboxy methyl cellulose, respectively. Two doses of Montelukast that is 10 mg/kg and 20 mg/kg[16 (link)17 (link)18 ] were used as a test drug and Enalapril 5 mg/kg was used as a positive control.[19 20 (link)]
Study procedure:[21 ] Forty Wistar rats of the required weight range were randomly allocated to four groups each group containing ten rats. The experimental study groups were: Vehicle control (STZ + Normal saline), Enalapril group (STZ + Enalapril 5 mg/kg), low-dose group (STZ + Montelukast 10 mg/kg) and high-dose group (STZ + Montelukast 20 mg/kg). The STZ-induced diabetic nephropathy model employed in this study had previously been standardised in identical lab settings.[19 ] Rats were kept fasting overnight in metabolic cages, the day before initiation of the study. Blood was collected from all rats from the retro-orbital plexus using non-heparinized micro-hematocrit capillary tubes and the samples were used for baseline estimation of fasting blood glucose (FBG), serum creatinine and blood urea nitrogen (BUN). Also, 24-hours urine samples were collected from metabolic cages to measure urine microalbumin levels. Then rats were housed according to the groups and the cages were labeled. On day 1, all rats were administered STZ 60 mg/kg single dose intraperitoneally and on day 7, FBG levels were assessed to confirm the induction of diabetes (levels above 150 mg/dL). The study drugs that is Montelukast and Enalapril were started as once-daily oral gavage using a rat-feeding needle from day 8 to day 42. At the end of the study, blood was collected from all the rats from the retro-orbital plexus to evaluate FBG, creatinine and BUN levels, and urine collection over 24-hours was done for urine microalbumin levels. After the collection of blood and urine samples, the rats were sacrificed. Exploratory laparotomy was performed and both the kidneys of the rats were dissected The kidneys were washed in cold saline and dried with the help of filter paper and immediately weighed on a digital weighing balance and the volume of the kidney was measured using the displacement method. One gram of kidney tissue was transferred to a glass bulb filled with 9 ml of cold phosphate buffer solution (PBS). The kidney tissue samples in the PBS glass bulbs were homogenised using homogenizer apparatus under constant motor speed. The entire process for homogenisation was carried out under strict cold chain control of the samples. Following the homogenisation, samples were processed for estimation of the kidney tissue malondialdehyde (MDA) and reduced glutathione (GSH) levels. The remaining second kidney was placed in a glass bulb filled with 10% neutral buffered formalin which was further processed for histopathological analysis.
Biochemical analysis: FBG, serum creatinine and BUN were assessed using an automated analyser. Urine microalbumin levels and MDA-GSH levels in kidney tissue were measured using double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method. MDA and GSH levels were measured to quantify the amount of oxidative stress induced by STZ. ELISA kits were purchased from Kinesis Dx, Los Angeles, USA.
Histopathological examination of kidneys: Kidneys in the formalin glass bulbs were sent to the laboratory for preparation of the slides and the slides were stained using haematoxylin and eosin. The slides were examined by a trained and experienced pathologist. Based on the current knowledge regarding histopathological changes that occur in STZ-induced diabetic nephropathy in rats, we devised a scoring system in consultation with the pathologist as shown in Table 1. This scoring was indirectly based on a study by Ozdemir O et al.[22 ]
Statistical analysis: Data from each study group were compiled and were expressed as Mean ± standard deviation (SD). The data were analysed using GraphPad InStat version 3.0. The level of significance was set at P < 0.05. Normality was checked by Shapiro-Wilk test. The study variables (except histopathological examination) were analysed using one-way ANOVA followed by post hoc Tukey’s test. The scoring of histopathological findings was expressed as median and range.