Manipal College of Pharmaceutical Sciences

Purpose of Review The rapid spread of virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has turned out to be a global emergency. Symptoms of this viral infection, coronavirus disease 2019 (COVID-19), include mild infections of the upper respiratory tract, viral pneumonia, respiratory failure, multiple organ failure and death. Till date, no drugs have been discovered to treat COVID-19 patients, and therefore, a considerable amount of interest has been shown in repurposing the existing drugs. Recent Findings Out of these drugs, chloroquine (CQ) and hydroxychloroquine (HCQ) have demonstrated positive results indicating a potential antiviral role against SARS-CoV-2. Its mechanism of action (MOA) includes the interference in the endocytic pathway, blockade of sialic acid receptors, restriction of pH mediated spike (S) protein cleavage at the angiotensin-converting enzyme 2 (ACE2) binding site and prevention of cytokine storm. Unfortunately, its adverse effects like gas...

As the incidence of COVID-19 increases with time, more and more efforts are made to pave a way out for the therapeutic strategies to deal with the disease progression. Inflammation being a significant influencer in COVID-19 patients, it drives our focus onto the signaling cascades of the JAK/STAT pathway. JAK phosphorylation mediated by cytokine receptor activation leads to phosphorylation of STATs that translocate into the nucleus to translate for inflammatory mediators. The SARS-CoV-2 structural proteins like spike, nucleocapsid, membrane and envelope proteins along with the non-structural proteins 1-16 including proteases like 3CL pro and PL pro promote its entry and survival in hosts. The SARS-CoV-2 infection triggers inflammation via the JAK/STAT pathway leading to recruitment of pneumocytes, endothelial cells, macrophages, monocytes, lymphocytes, natural killer cells and dendritic cells progressing towards cytokine storm. This produces various inflammatory markers in the host that determine the disease severity. The JAK/STAT signaling also mediates immune responses via B cell and T cell differentiation.With an attempt to reduce excessive inflammation, JAK/STAT inhibitors like Ruxolitinib, Baricitinib, Tofacitinib have been employed that mediate its actions via suppressors of cytokine signaling, cytokine inducible SH2 containing protein, Protein inhibitor of activated STAT and protein tyrosine phosphatases. Even though they are implicated with multiple adverse effects, the regulatory authorities have supported its use, and numerous clinical trials are in progress to prove their safety and efficacy. On the contrary, the exact mechanism of JAK/STAT inhibition at molecular levels remains speculative for which further investigations are required.

What began with a sign of pneumonia-related respiratory disorders in China has now become a pandemic named by WHO as Covid-19 known to be caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The SARS-CoV-2 are newly emerged b coronaviruses belonging to the Coronaviridae family. SARS-CoV-2 has a positive viral RNA genome expressing open reading frames that code for structural and non-structural proteins. The structural proteins include spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins. The S1 subunit of S protein facilitates ACE2 mediated virus attachment while S2 subunit promotes membrane fusion. The presence of glutamine, asparagine, leucine, phenylalanine and serine amino acids in SARS-CoV-2 enhances ACE2 binding. The N protein is composed of a serine-rich linker region sandwiched between N Terminal Domain (NTD) and C Terminal Domain (CTD). These terminals play a role in viral entry and its processing post entry. The NTD forms orthorhombic crystals and binds to the viral genome. The linker region contains phosphorylation sites that regulate its functioning. The CTD promotes nucleocapsid formation. The E protein contains a NTD, hydrophobic domain and CTD which form viroporins needed for viral assembly. The M protein possesses hydrophilic C terminal and amphipathic N terminal. Its long-form promotes spike incorporations and the interaction with E facilitates virion production. As each protein is essential in viral functioning, this review describes the insights of SARS-CoV-2 structural proteins that would help in developing therapeutic strategies by targeting each protein to curb the rapidly growing pandemic.

p-Coumaric acid is a natural hydroxycinnamic acid existing in grapes and wine. It is the precursor of the 4-ethylphenol molecule through the bioconversion reaction by Brettanomyces yeast. Chromatographic methods are the most common techniques to detect p-coumaric acid. It is known that this acid is highly unstable in analysis and fermentation experiments. This paper highlights the problems occurring in p-coumaric acid analysis in wine fermentation conditions when studying its bioconversion. First, it was shown that p-coumaric acid was unstable at elevated temperature. On the other hand, it was found that in our experimental conditions p-coumaric acid reacted with ethanol. This work revealed also that the p-coumaric acid is partially adsorbed on Brettanomyces yeast, certainly on cell walls. Because of these phenomena the quantity of p-coumaric acid which can participate to the bioconversion into ethylphenol decreases.

This study demonstrates cytotoxic and genotoxic potential of juglone, a chief constituent of walnut, and its underlying mechanisms against melanoma cells. MTT assay and clonogenic assay were used to study cytotoxicity, micronucleus assay to assess genotoxicity, glutathione (GSH) assay and 2 0 ,7 0 -dicholorofluorescein diacetate (DCFH-DA) assay to evaluate the oxidative stress induction. Apoptosis/necrosis induction was analysed by flow cytometry. We observed a concentration-dependent decrease in cell survival with a corresponding increase in the lactate dehydrogenase levels. A dose-dependent increase in the frequency of micronucleated binucleate cells indicated the potential of juglone to induce cytogenetic damage in melanoma tumor cells. Moreover, results of the micronuclei study indicated division delay in the proliferating cell population by showing decrease in the cytokinesis blocked proliferation index. Further, juglone-induced apoptosis and necrosis could be demonstrated by oligonucleosomal ladder formation, microscopic analysis, increase in the hypodiploid fraction (sub Go peak in DNA histogram), as well as an increased percentage of AnnexinV(þ)/PI(þ) cells detected by flow cytometry. A significant concentration-dependent decrease in the glutathione levels and increase in dichlorofluorescein (DCF) fluorescence after juglone treatment confirmed the ability of juglone to generate intracellular reactive oxygen species. The cytotoxic effect of juglone can be attributed to mechanisms including the induction of oxidative stress, cell membrane damage, and a clastogenic action leading to cell death by both apoptosis and necrosis. Ó

Quinones have diverse pharmacological properties including antibacterial, antifungal, antiviral, antiinflammatory, antipyretic and anticancer activity. The cytotoxic potential of 1,4-naphthoquinone (NQ14) was studied against B16F1 melanoma cells grown in vitro. NQ14 treatment resulted in a concentration-dependent cytotoxicity as indicated by MTT assay and lactate dehydrogenase leakage assay. Depletion in cellular glutathione levels after 1 h incubation with NQ14 correlated with the corresponding increase in reactive oxygen species generation as determined by 2 0 ,7 0 -dicholorofluorescein diacetate assay suggests the role of oxidative stress in cell death. The frequency of micronucleated binucleate cells increased with increasing doses of NQ14 with a corresponding decrease in the cytokinesis block proliferation index indicating the drug induced genotoxicity and cell division delay. Further, a dose-dependent decrease in the clonogenic cell survival indicated the potential of NQ14 to inhibit cell proliferation contributing to cell death. The cell death after NQ14 treatment may be attributed to apoptosis as seen in DNA ladder pattern along with necrosis as indicated in flow cytometric analysis of Annexin V/PI stained cells. Results of the present study demonstrate the cytotoxic and genotoxic potential of NQ14 by the induction of oxidative stress mediated mechanisms leading to tumor cell kill.

This study demonstrates cytotoxic and genotoxic potential of juglone, a chief constituent of walnut, and its underlying mechanisms against melanoma cells. MTT assay and clonogenic assay were used to study cytotoxicity, micronucleus assay to assess genotoxicity, glutathione (GSH) assay and 2 0 ,7 0 -dicholorofluorescein diacetate (DCFH-DA) assay to evaluate the oxidative stress induction. Apoptosis/necrosis induction was analysed by flow cytometry. We observed a concentration-dependent decrease in cell survival with a corresponding increase in the lactate dehydrogenase levels. A dose-dependent increase in the frequency of micronucleated binucleate cells indicated the potential of juglone to induce cytogenetic damage in melanoma tumor cells. Moreover, results of the micronuclei study indicated division delay in the proliferating cell population by showing decrease in the cytokinesis blocked proliferation index. Further, juglone-induced apoptosis and necrosis could be demonstrated by oligonucleosomal ladder formation, microscopic analysis, increase in the hypodiploid fraction (sub Go peak in DNA histogram), as well as an increased percentage of AnnexinV(þ)/PI(þ) cells detected by flow cytometry. A significant concentration-dependent decrease in the glutathione levels and increase in dichlorofluorescein (DCF) fluorescence after juglone treatment confirmed the ability of juglone to generate intracellular reactive oxygen species. The cytotoxic effect of juglone can be attributed to mechanisms including the induction of oxidative stress, cell membrane damage, and a clastogenic action leading to cell death by both apoptosis and necrosis. Ó

Quinones have diverse pharmacological properties including antibacterial, antifungal, antiviral, antiinflammatory, antipyretic and anticancer activity. The cytotoxic potential of 1,4-naphthoquinone (NQ14) was studied against B16F1 melanoma cells grown in vitro. NQ14 treatment resulted in a concentration-dependent cytotoxicity as indicated by MTT assay and lactate dehydrogenase leakage assay. Depletion in cellular glutathione levels after 1 h incubation with NQ14 correlated with the corresponding increase in reactive oxygen species generation as determined by 2 0 ,7 0 -dicholorofluorescein diacetate assay suggests the role of oxidative stress in cell death. The frequency of micronucleated binucleate cells increased with increasing doses of NQ14 with a corresponding decrease in the cytokinesis block proliferation index indicating the drug induced genotoxicity and cell division delay. Further, a dose-dependent decrease in the clonogenic cell survival indicated the potential of NQ14 to inhibit cell proliferation contributing to cell death. The cell death after NQ14 treatment may be attributed to apoptosis as seen in DNA ladder pattern along with necrosis as indicated in flow cytometric analysis of Annexin V/PI stained cells. Results of the present study demonstrate the cytotoxic and genotoxic potential of NQ14 by the induction of oxidative stress mediated mechanisms leading to tumor cell kill.

The objective of this investigation was to develop an injectable, depot-forming drug delivery system for insulin based on microparticle technology to maintain constant plasma drug concentrations over prolonged period of time for the effective control blood sugar levels. Formulations were optimized with two well-characterized biodegradable polymers namely, poly(DL-lactide-co-glycolide) and poly-e-caprolactone and evaluated in vitro for physicochemical characteristics, drug release in phosphate buffered saline (pH 7.4), and evaluated in vivo in streptozotocin-induced hypoglycemic rats. With a large volume of internal aqueous phase during w/o/w double emulsion solvent evaporation process and high molecular weight of the polymers used, we could not achieve high drug capture and precise control over subsequent release within the study period of 60 days. However, this investigation revealed that upon subcutaneous injection, the biodegradable depot-forming polymeric microspheres controlled the drug release and plasma sugar levels more efficiently than plain insulin injection. Preliminary pharmacokinetic evaluation exhibited steady plasma insulin concentration during the study period. These formulations, with their reduced frequency of administration and better control over drug disposition, may provide an economic benefit to the user compared with products currently available for diabetes control.

The influence of ultrasound on percutaneous absorption of ketorolac tromethamine was studied in vitro across rat skin. Sonication was carried out with a continuous mode, at an intensity of 1-3 W/cm 2 and a frequency of 1 MHz for 30 min. A significant increase in permeation of ketorolac through rat skin was observed with the applied sonication at 3 W/cm 2 when compared with permeation at 1 and 2 W/cm 2 . Enhanced ketorolac penetration at 3 W/cm 2 can be explained by the mechanical and/or thermal action of ultrasound waves. The distance of the ultrasound probe from the skin surface did not influence the flux of the drug. Pretreatment of skin by 5% d-limonene in ethanol for 2 hr followed by sonication at 3 W/cm 2 (30 min) significantly enhanced the permeation of ketorolac when compared with passive flux with or without enhancer pretreatment.

In the present investigation, a poly(dl-co-glycolide) (PLGA)-based, microspheric depot system for bleomycin (BLM) has been formulated, and the same has been evaluated in-vivo in C57BL/6J mice bearing transplantable melanoma B16F1 murine solid tumour. The microparticulate delivery systems were formulated employing a water-in-oil-in-water (W/O/W) emulsion-solvent evaporation technique and characterized in-vitro. The microspheres were injected subcutaneously to form a drug depot at the site of injection in mice bearing experimental tumours and the drug was continuously infused into the systemic circulation with progressive biodegradation. The drug-loaded microspheres exhibited improved pharmacodynamic e cacy, as evidenced by retarded tumour growth kinetics. Preliminary pharmacokinetic studies illustrated controlled release of the drug into the systemic circulation over the study period to exert an anti-neoplastic action. These studies demonstrated the feasibility of employing a PLGA-based microparticulate system as an e ective biodegradable, injectable, depot-forming therapeutic system for long-term administration of anti-neoplastic agents.

The antitumor and radiosensitizing effects of withaferin A (WA), a steroidal lactone from Withania somnifera, was studied on Ehrlich ascites carcinoma in vivo. The acute LD50(14) for WA in Swiss mice was -80 mg/kg. Twenty-four hours after i.p. inoculation of lo6 tumor cells, WA was injected i.p. at different dose fractions (5 or 7.5 mg/kg x 8, 10 mg/kg x 5, 20 or 30 mg/kg x 2) with or without abdominal gamma irradiation (RT, 7.5 Gy) after the first drug dose. Increase in life span and tumor-free survival were studied up to 120 days. The drug inhibited tumor growth and increased survival, which was dependent on the WA dose per fraction rather than the total dose. Combination of RT with all the drug schedules increased tumor cure and tumor-free survival, the best effect seen after 2 fractions of 30 mg/kg each. In another experiment WA was given as 2 (40 mg/kg x 2), 3 (30 mg/ kg x 3) or 4 (20 mg/kg x 4) fractions at 5, 7 or 10 days after tumor inoculation with or without RT after the first drug dose. At 7 and 10 days after inoculation the drug was effective only at 40 mg/kg x 2, but with RT 30 mg/kg x 3 produced an equal effect (20Y0 survival) on 7 day old tumors.

Dianex, a polyherbal formulation consisting of the aqueous extracts of Gymnema sylvestre, Eugenia jambolana, Momordica charantia Azadirachta indica, Cassia auriculata, Aegle marmelose, Withania somnifera and Curcuma longa was screened for hypoglycemic activity in normal and streptozotocin induced diabetic mice. Dianex was administered in different doses of 100–500 mg/kg/day orally in acute (6 h) and long-term (6 weeks) studies. Blood glucose levels were checked 2–6 h after treatment in acute studies and every 2 weeks in long-term studies. Body weight was recorded on the first and final day of the treatment in the long-term studies with diabetic mice. After 6 weeks, high-density lipoprotein, triglycerides, total cholesterol, alanine transaminase (ALT), aspertate transaminase (AST), urea and creatinine were estimated in serum of the diabetic mice. Glycogen and total protein levels were estimated in the liver. Also, the liver and pancreas was subjected to histological examination. Oral glucose tolerance and in vitro free radical scavenging activity was also studied.Dianex produced significant (p < 0.05) hypoglycemic activity at 250–500 mg/kg doses in both normal and diabetic mice in acute and long-term studies. The body weight of diabetic mice significantly (p < 0.05) increased with all tested doses of Dianex. The elevated triglycerides, cholesterol, ALT, AST, urea and creatinine levels in diabetic mice were significantly (p < 0.05) reduced at the doses of 250 and 500 mg/kg. The liver glycogen and protein levels were both significantly (p < 0.05) increased in diabetic mice at 250 and 500 mg/kg doses. Dianex increased the glucose tolerance significantly (p < 0.05) in both normal and diabetic mice at all the doses tested. Histopathological examination showed that the formulation decreased streptozotocin induced injury to the tissues at all the doses tested. It produced significant (p < 0.05) free radical scavenging activity against ABTS+, DPPH and hydroxyl free radicals at the concentrations ranging between 10–1000 µg/ml.Thus, in the present study, Dianex produced significant hypoglycemic activity in both normal and diabetic animals. It also reversed other diabetic complications in diabetic mice at 250 and 500 mg/kg doses. In our earlier study, Dianex was well tolerated in laboratory animals at higher doses (upto 10 g/kg in mice, acute toxicity; upto 2.5 g/kg in rats, subacute toxicity studies for 30 days) without exhibiting any toxic manifestation. Hence, Dianex may be useful in the treatment of diabetes mellitus. Copyright © 2005 John Wiley & Sons, Ltd.

Methotrexate was encapsulated in "niosomes" of sorbitan monostearate (Span 60) by the reverse phase evaporation technique. After i.v. infusion into mice methotrexate concentration was higher in all tissues except kidney with niosomal methotrexate than with methotrexate solution. Niosome encapsulation and macrophage activation enhanced the delivery of methotrexate to the tumor and increased its antitumor activity. @ 1983 Academic P r e~, Inc.

The objective of the present study was to formulate insulin gel for intranasal administration and to evaluate with respect to in vitro release studies and hypoglycemic activity in animal model and healthy human volunteers. The insulin gel was formulated using the combination of carbopol and hydroxypropyl methylcellulose as gelling agent. The in vivo efficacy of insulin gel administered intranasally was assessed by measuring the blood glucose levels and serum insulin levels at specified time intervals in rats and humans. The use of bioadhesive nasal gel containing insulin not only promoted the prolonged contact between the drug and the absorptive sites in the nasal cavity but also facilitated direct absorption of medicament through the nasal mucosa. Absorption of the drug through the nasal mucosa was high in the first 0.5 to 1.5 hours of the study with a sharp decline in blood sugar and rise in insulin values corresponding to that decline in blood sugar. This study further demonstrates that administration of insulin intranasally in gel form is a pleasant and painless alternative to injectable insulin.

Methotrexate was encapsulated i n niosomes prepared using Tweens and Spans. The s i z e d i s t r i b u t i o n , entrapment e f f i c i e n c y , pharrnacokinetics and e f f e c t on tumour remission o f mice transplanted w i t h S-180 Sarcoma were evaluated. Niosomes prepared w i t h Span 60 gave promising r e s u l t s .

PURPOSE: The objective of the present study was to develop the membrane controlled transdermal systems of glibenclamide and to evaluate with respect o various in vitro and in vivo parameters. METHODS: The membrane moderated transdermal systems were prepared using drug containing carbopol gel as reservoir and ethyl cellulose, Eudragit RS-100, Eudragit RL-100 and Ethylene vinyl acetate (EVA) (2%, 9% and 19% vinyl acetate content) rate controlling membranes. The possible interaction between drug and polymer was studied by IR spectroscopy, DSC and HPTLC analysis. The formulations were subjected to various physicochemical studies, in vitro drug release studies and permeation studies through mouse skin. The blood glucose reducing hypoglycemic activity of the systems was studied in both normal and diabetic mice. Various biochemical parameters and histopathological studies were carried out after treating the diabetic mice for 6 weeks. Skin irritation tests, oral glucose tolerance test and pharmacokinetic evaluations were carried out in mice. RESULTS: The results suggested no interaction between drug and polymer. Variations in drug release/permeation profiles among the formulations containing different rate controlling membranes were observed. The scanning electron microscopic studies of EVA membranes demonstrated no changes in the surface morphology after in vitro skin permeation studies. The system with EVA rate controlling membrane (with 19% vinyl acetate) was selected for in vivo experiments. The transdermal system produced better improvement with respect to hypoglycemic activity, glucose tolerance test, all the tested biochemical, histopathological and pharmacokinetic parameters compared to oral administration, and exhibited negligible skin irritation. CONCLUSION: The present study shows that membrane controlled transdermal systems of glibenclamide exhibited better control of hyperglycemia and more effectively reversed the diabetes mellitus complications than oral glibenclamide administration in mice.