Usage Pattern of Fixed-dose Combinations at ICMR Network of Rational Use of Medicine Centres across India: Recommendations for Policymakers and Prescribers.

AIM: Irrational use of medicines is a global problem. In India, one contributing factor is the availability of a large number of fixed-dose combinations (FDCs). To improve rational use and to strengthen policies, it is important to assess the usage patterns and rationality of FDCs. METHODS: This study was conducted as part of a 1-year prospective cross-sectional analysis of prescriptions in the outpatient clinics of broad specialities from 13 tertiary care hospitals across India. Five most commonly prescribed FDCs in each center were analyzed. In addition, all the prescribed FDCs were classified as per the Kokate Committee classification and it was noted whether any of the FDCs were irrational or banned as per the reference lists released by regulatory authorities. RESULTS: A total of 4,838 prescriptions were analyzed. Of these, 2,093 (43.3%) prescriptions had at least one FDC. These 2,093 prescriptions had 366 different FDCs. Of the 366 FDCs, 241 were rational; 10 were irrational; 14 required further data generation; and the remaining 96 FDCs could not be categorized into any of the above. Vitamins and minerals/supplements, antibacterial for systemic use, and drugs for gastroesophageal reflux disease (GERD) and peptic ulcer were the most used FDCs. CONCLUSION: Based on the finding that some prescriptions contained irrational FDCs, it is recommended that a rigorous, regular, and uniform method of evaluation be implemented to approve/ban FDCs and that prescribers be periodically notified about the status of the bans.

An observational multi-centric COVID-19 sequelae study among health care workers.

Background: India has seen more than 43 million confirmed cases of COVID-19 as of April 2022, with a recovery rate of 98.8%, resulting in a large section of the population including the healthcare workers (HCWs), susceptible to develop post COVID sequelae. This study was carried out to assess the nature and prevalence of medical sequelae following COVID-19 infection, and risk factors, if any. Methods: This was an observational, multicenter cross-sectional study conducted at eight tertiary care centers. The consenting participants were HCWs between 12 and 52 weeks post discharge after COVID-19 infection. Data on demographics, medical history, clinical features of COVID-19 and various symptoms of COVID sequelae was collected through specific questionnaire. Finding: Mean age of the 679 eligible participants was 31.49 ± 9.54 years. The overall prevalence of COVID sequelae was 30.34%, with fatigue (11.5%) being the most common followed by insomnia (8.5%), difficulty in breathing during activity (6%) and pain in joints (5%). The odds of having any sequelae were significantly higher among participants who had moderate to severe COVID-19 (OR 6.51; 95% CI 3.46-12.23) and lower among males (OR 0.55; 95% CI 0.39-0.76). Besides these, other predictors for having sequelae were age (≥45 years), presence of any comorbidity (especially hypertension and asthma), category of HCW (non-doctors vs doctors) and hospitalisation due to COVID-19. Interpretation: Approximately one-third of the participants experienced COVID sequelae. Severity of COVID illness, female gender, advanced age, co-morbidity were significant risk factors for COVID sequelae. Funding: This work is a part of Indian Council for Medical Research (ICMR)- Rational Use of Medicines network. No additional financial support was received from ICMR to carry out the work, for study materials, medical writing, and APC.

Use of drugs not listed in the National List of Essential Medicines: Findings from a prescription analysis by the Indian Council of Medical Research-Rational Use of Medicines Centres Network in tertiary care hospitals across India.

BACKGROUND: The concept of listing essential medicines can lead to improved supply and access, more rational prescribing, and lower costs of drugs. However, these benefits hinge on the prescription of drugs from an Essential Medicines List (EML). Several studies have highlighted the problem of underutilization of EMLs by prescribers. Therefore, as part of prescription research by the Indian Council of Medical Research-Rational Use of Medicines Centres Network, we evaluated the extent of prescription of drugs not listed in the National List of Essential Medicines (NLEM). MATERIALS AND METHODS: Prescriptions of outpatients from participating centers were included after obtaining verbal/written informed consent as approved by the Ethics Committee, and evaluated for prescription of drugs from the NLEM 2015. RESULTS: Analysis of 4838 prescriptions from 13 tertiary health-care institutes revealed that 2677 (55.33%) prescriptions had at least one non-NLEM drug prescribed. In all, 5215 (31.12%) of the total 16,758 drugs prescribed were not in NLEM. Of these, 2722 (16.24%) were single drugs and 2493 (14.88%) were fixed-dose combinations (FDCs). These comprised 700 different drug products - 346 single drugs and 354 FDCs. The average number of non-NLEM drugs prescribed per prescription was 1.08, while the average number of all drugs prescribed was 3.35 per prescription. It was also found that some of the non-NLEM drugs prescribed had the potential to result in increased cost (for example, levocetirizine), increased adverse effects (dextromethorphan), and less effectiveness (losartan) when compared to their NLEM counterparts. Nonavailability of an essential drug (oral hydroxocobalamin) was another important finding of our study. CONCLUSION: This study highlights the extent and pattern of drugs prescribed from outside the NLEM at the tertiary health-care level and the need for training and enhanced awareness among prescribers for greater utilization of the NLEM.

In vitro Anti-Proliferative Effect of Tephrosia purpurea on Human Hepatocellular Carcinoma Cells.

BACKGROUND: Tephrosia purpurea is an Indian herb used in traditional medicine to treat various diseases such as jaundice, asthma, liver and urinary disorders. However, the anti-cancer potential of T. purpurea on hepatocellular carcinoma (HCC) is poorly understood. Therefore, this study aims to investigate the anti-cancer activity of T. purpurea in HepG2 hepatocellular carcinoma cells. METHODS: The leaves and root of T. purpurea were extracted with methanol using soxhlet apparatus. The cytotoxicity of the T. purpurea extracts in HepG2 cells was evaluated using MTT assay whereas the mode of cell death was examined by AOEB, Hoechst and JC1 staining under a fluorescence microscope. T. purpurea extracts-induced caspase-3 expression was investigated using colorimetric assay. RESULTS: The leaves and root extracts inhibited HepG2 cell growth at the IC50 of 102.33 ± 10.26 µg/mL and 276.67 ± 20.43 µg/mL respectively at 24 h. Chromatin condensation, nuclear fragmentation, apoptotic bodies formation and mitochondrial membrane depolarization were observed in HepG2 cells treated with both extracts. The caspase-3 expression was significantly (p < 0.05) increased in extracts treated cells when compared to control. CONCLUSION: The leaves and root extracts of T. purpurea induce apoptosis mediated cell death in HepG2 cells. SUMMARY: The leaves and root extracts of T. purpurea exhibited anticancer activity in HepG2 hepatocellular carcinoma cells. These extracts induced cell shrinkage, DNA condensation and fragmentation, mitochondrial membrane depolarization and upregulated caspase-3 expression indicating T. purpurea extracts induce apoptosis in HepG2 cells. Abbreviation used: AO: acridine orange, DMSO: dimethyl sulfoxide, EB: ethidium bromide, IC50: the concentration at which 50% of cancer cells are dead, JC-1: 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide, MTT: 3-4, 5-dimethylthiazole-2-yl, 2,5-diphenyl tetrazolium bromide, PBS: phosphate-buffered saline, ΔΨm: mitochondrial trans-membrane potential.

In vitro antioxidant and cytotoxic potential of different parts of Tephrosia purpurea.

The antioxidant and cytotoxic properties of four major parts of methanolic extracts of Tephrosia purpurea including leaves, root, stem and seed were investigated and compared. In vitro antioxidant activity of T. purpurea extracts was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), reducing power assay and antihemolytic assay. In vitro cytotoxic effect of T. purpurea extracts on SW620 colorectal cancer cell line was studied using 3-(4, 5-dimethylthiazolyl -2,5-diphenyl-tetrazolium bromide (MTT) assay. Folin-ciocalteu and aluminium chloride methods were used to determine the total phenolic and flavonoid contents respectively. Among the four extracts studied, leaves extract showed the highest antioxidant activity, DPPH: 186.3 ± 14.0 µg/mL, FRAP: 754.2 ± 50.9 µmol Fe(II)/mg and reducing power activity: 65.7 ± 4.2 µg/mg of quercetin equivalent (QE/mg) and there was no significant difference observed in antihemolytic activity. Leaves extract showed effective cytotoxicity on colorectal cancer cells (IC50: 95.73 ± 9.6 µg/mL) and also had the higher total phenolic (90.5 ± 6.7 µg/mg of gallic acid equivalent (GAE/mg) and flavonoid content (21.8 ± 5.4 µg QE/mg). These results suggest higher antioxidant and cytotoxic activities of leaves extract in comparison with other extracts and these activities could be due to the presence of rich phenolic and flavonoid content.

Evaluation of in vivo antitumor activity of cleistanthin B in Swiss albino mice.

To evaluate the in vivo antitumor activity of cleistanthin B in Ehrlich's ascites carcinoma (EAC) and Dalton's ascites lymphoma (DAL) cell lines induced malignant ascites mouse models and DAL cell line induced solid tumor mouse model. All animals were injected with 2 × 106 EAC/DAL cells i.p./s.c. to induce malignant ascites and solid tumor and treated with 5-fluorouracil (5-FU) 20 mg/kg or cleistanthin B for 10 days. Cleistanthin B was given at three doses viz. 25, 50 and 100 mg/kg. The percentage increase in life span and the overall survival in malignant ascites animals and the tumor volume in solid tumor animals were measured. The haematological parameters were assessed in all animals before and 2 weeks after the treatment. Cleistanthin B 50 mg/kg and 5-FU significantly prolonged the life span (>25%) of malignant ascites tumor bearing animals. The overall survival was significantly improved by both. Only cleistanthin B 50 mg/kg significantly reduced the elevated WBC counts in EAC tumor bearing animals. Both 5-FU and cleistanthin B 50 mg/kg reversed the malignancy induced increase in neutrophils and platelet counts and decrease in lymphocyte counts but not to the normal range. Only 5-FU significantly reduced the solid tumor volume. None of the three doses of cleistanthin B was effective against the solid tumor. Cleistanthin B has antitumor activity against EAC and DAL tumor mice but it is not as effective as 5-FU. At 50 mg/kg dose cleistanthin B exerts significant antitumor activity compared to 25 and 100 mg/kg dose. Its effect on WBC count is higher and advantageous when compared to 5-FU. But cleistanthin B in the doses used is not effective against solid tumor.

Hypertension influences the exponential progression of inflammation and oxidative stress in streptozotocin-induced diabetic kidney.

OBJECTIVE: To investigate the association of hypertension coexisting with diabetes mellitus with oxidative stress and inflammation in the kidneys of streptozotocin (STZ)-induced diabetic rats. MATERIALS AND METHODS: Male Wistar rats were used for the experiments. Blood glucose (BG), urea, blood pressure (BP), and heart rate (HR) were analyzed before and 48 h after STZ injection. Further, these parameters were monitored up to 3 months of diabetes induction. Subsequently, the inflammatory markers (C-reactive protein, tumor necrosis factor-alpha, and nitrate) and oxidative stress markers were estimated after 3 months of diabetes induction in the kidney homogenate. Histological analysis of renal tissue was also carried out. RESULTS: Linear elevation of BG, urea, mean arterial pressure (MAP), and HR was observed up to 3 months of diabetes induction. In the same manner, inflammatory and oxidative stress markers were also found to be significantly increased. Notably, the histological analysis revealed the signs of nephropathy such as increased mesangial cell number, thickness of basement membrane, and renal artery. Inflammatory and oxidative stress markers positively correlated with elevated BP and BG, but the correlation was better with BP rather than BG. CONCLUSION: Hypertension has a strong implication in the increased oxidative stress and inflammation of diabetic kidney at the very early stage of diabetes mellitus.

Identification of natural inhibitors against angiotensin I converting enzyme for cardiac safety using induced fit docking and MM-GBSA studies.

BACKGROUND: Cleistanthins A and B are isolated compounds from the leaves of Cleistanthus collinus Roxb (Euphorbiaceae). This plant is poisonous in nature which causes cardiovascular abnormalities such as hypotension, nonspecific ST-T changes and QTc prolongation. The biological activity predictions spectra of the compounds show the presence of antihypertensive, diuretic and antitumor activities. OBJECTIVE: Objective of the present study was to determine the in silico molecular interaction of cleistanthins A and B with Angiotensin I- Converting Enzyme (ACE-I) using Induced Fit Docking (IFD) protocols. MATERIALS AND METHODS: All the molecular modeling calculations like IFD docking, binding free energy calculation and ADME/Tox were carried out using Glide software (Schrödinger LLC 2009, USA) in CentOS EL-5 workstation. RESULTS: The IFD complexes showed favorable docking score, glide energy, glide emodel, hydrogen bond and hydrophobic interactions between the active site residues of ACE-I and the compounds. Binding free energy was calculated for the IFD complexes using Prime MM-GBSA method. The conformational changes induced by the inhibitor at the active site of ACE-I were observed based on changes of the back bone Cα atoms and side-chain chi (x) angles. The various physicochemical properties were calculated for these compounds. Both cleistanthins A and B showed better docking score, glide energy and glide emodel when compared to captopril inhibitor. CONCLUSION: These compounds have successively satisfied all the in silico parameters and seem to be potent inhibitors of ACE-I and potential candidates for hypertension.

Sub-chronic toxicological evaluation of cleistanthin A and cleistanthin B from the leaves of Cleistanthus collinus (Roxb.).

OBJECTIVE: To investigate the toxicological effects of cleistanthin A and cleistanthin B using sub-chronic toxicity testing in rodents. METHOD: Cleistanthins A and B were isolated from the leaves of Cleistanthus collinus. Both the compounds were administered orally for 90 days at the concentration of 12.5, 25 and 50 mg/kg, and the effects on blood pressure, biochemical parameters and histology were assessed. The dose for sub-chronic toxicology was determined by fixed dose method according to OECD guidelines. RESULT: Sub-chronic toxicity study of cleistanthins A and B spanning over 90 days at the dose levels of 12.5, 25 and 50 mg/kg (once daily, per oral) revealed a significant dose dependant toxic effect in lungs. The compounds did not have any effect on the growth of the rats. The food and water intake of the animals were also not affected by both cleistanthins A and B. Both the compounds did not have any significant effect on liver and renal markers. The histopathological analysis of both cleistanthins A and B showed dose dependent morphological changes in the brain, heart, lung, liver and kidney. When compared to cleistanthin A, cleistanthin B had more toxic effect in Wistar rats. Both the compounds have produced a dose dependent increase of corpora amylacea in brain and induced acute tubular necrosis in kidneys. In addition, cleistanthin B caused spotty necrosis of liver in higher doses. CONCLUSION: The present study concludes that both cleistanthin A and cleistanthin B exert severe toxic effects on lungs, brain, liver, heart and kidneys. They do not cause any significant pathological change in the reproductive system; neither do they induce neurodegenerative changes in brain. When compared to cleistanthin A, cleistanthin B is more toxic in rats.

Biodistribution properties of cleistanthin A and cleistanthin B using magnetic resonance imaging in a normal and tumoric animal model.

AIM: To determine the biodistribution properties of cleistanthin A and cleistanthin B in rodents using magnetic resonance imaging (MRI). MATERIALS AND METHODS: Cleistanthins A and B, constituents of Cleistanthus collinus Roxb., were labelled with gadolinium (Gd(3+)) directly and injected into normal and tumoric nude mice. The tissue signal intensity was measured using MRI to perform a noninvasive kinetic assay. Wistar rats were used for determination of the grayscale intensity to observe the distribution patterns of of cleistanthins A and B. RESULTS: Cleistanthin A is kinetically more attractive to the gastrointestinal tract than is cleistanthin B, which gets accumulated in muscular tissues of mice in greater concentrations compared with cleistanthin A. Cleistanthin B but not cleistanthin A showed tumoric affinity and exhibited a tumor kinetic attraction in tumoric mice. In rats, cleistanthin A showed greater grayscale intensities in the brain, liver, and skeletal muscles in immediate post contrast MRI images, whereas the gadolinium tagged cleistanthin B showed higher grayscale intensities in the cardiac muscle and skeletal muscles in delayed post contrast MRI images. CONCLUSIONS: Cleistanthin A is more pharmacokinetically attractive to the gastrointestinal tract than cleistanthin B.

Measurement of invasive blood pressure in rats.

Blood pressure (BP) is one of the vital parameters used to assess the cardiovascular functions of a mammal. BP is commonly recorded using invasive, noninvasive, and radio telemetry methods, but invasive blood pressure (IBP) recording is considered the gold standard. IBP provides a direct indication of the effect of the investigational products on the circulatory system. Recording the IBP in rodents is an essential part of the preliminary screening of any product to determine its effect on the cardiovascular system. The present article describes the measurement of the IBP in Wistar rats/Sprague Dawley rats.

Molecular docking and ex vivo pharmacological evaluation of constituents of the leaves of Cleistanthus collinus (Roxb.) (Euphorbiaceae).

OBJECTIVE: To investigate the involvement of alpha adrenergic receptors in hypotension induced by cleistanthin A and cleistanthin B. MATERIALS AND METHODS: Cleistanthins A and B were isolated from the leaves of Cleistanthus collinus using a column chromatographic method and purified. Structures were confirmed by spectroscopic analysis. The compounds were prepared for molecular docking studies using Ligprep 2.3 module and Induced Fit Docking was carried out against α-1 adrenergic receptors using Glide. The ex vivo experiments were carried out on male Wistar rats. Under anaesthesia, the femoral vein and carotid artery were cannulated for drug administration and for monitoring the blood pressure, respectively. The effect of epinephrine, norepinephrine, acetylcholine, histamine and dopamine were recorded before and after the administration of cleistanthin A or cleistanthin B. The molecular docking studies showed favorable molecular interactions, glide score, energy and emodel. RESULT: Cleistanthins A and B per se reduced the mean blood pressure and the effect was dose dependent. Both the compounds reduced the effect of epinephrine, norepinephrine and α-1 receptor activity of dopamine. Cleistanthin B significantly increased the duration of action of acetylcholine on mean blood pressure. CONCLUSION: The molecular docking and ex vivo studies conclude that cleistanthin A and cleistanthin B have significant α-1 adrenergic receptor antagonist effect on the peripheral vascular system.

Effects of cleistanthins A and B on blood pressure and electrocardiogram in Wistar rats.

We have studied the effects of cleistanthin A and cleistanthin B, phytoconstituents isolated from the leaves of Cleistanthus collinus Roxb. (Euphorbiaceae), on blood pressure, electrocardiogram, and barium chloride-induced arrhythmia in Wistar rats. The two compounds were isolated by column chromatography and their identity was confirmed spectroscopically. A healthy, male Wistar rat was used to record the invasive blood pressure and electrocardiograph. The antiarrhythmic effects of cleistanthins A and B were studied using the barium chloride model. Both cleistanthin A and cleistanthin B showed a dose-dependent hypotensive effect. Both compounds reduced the mean blood pressure significantly although the dose required for the effect was higher in the case of cleistanthin B. In the electrocardiogram, cleistanthins A and B significantly altered the electrical activity of the heart, the changes were transient and of no further consequence. Intravenous injection of 64 microg or more of cleistanthins A and B caused a sudden respiratory depression without affecting the electrocardiogram. Cleistanthins A and B did not display any antiarrhythmic effect against barium chloride-induced arrhythmia. In conclusion, both cleistanthin A and cleistanthin B exert a hypotensive effect and have no antiarrhythmic effect against barium chloride-induced arrhythmia in Wistar rats.