The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, resulted in global health and economic crisis at an exceptional level. The high transmissibility of SARS-CoV-2, a lack of population immunity, and the prevalence of severe clinical outcomes created a need for the rapid development of effective therapeutic countermeasures. Sybodies, or synthetic nanobodies, are a novel and unique class of synthetic antigen-binding fragments ideal for large-scale production. We created a neutralizing sybody directed against the receptor-binding domain (RBD) epitope of the Spike protein of SARS-CoV-2 and conjugated it to remdesivir to create nanobody-drug-conjugate (NDC). We used a mouse model of infection to determine the capacity of the NDC to reduce disease severity and mortality. K18-hACE2 mice treated with NDC prior to, simultaneously with, and/or post-SARS-CoV-2 infection had reduced weight loss, mortality, lung pathology, and viral RNA in their lungs. This protection was found to be dependent on the protein structure of the sybody. Delivery of these novel therapeutic NDCs through nasal inhalation using a nebulizer could offer a convenient method for patients to self-administer treatments or as a prophylaxis. This platform therapy could apply to new variants of the ongoing SARS-CoV-2 epidemics, or to other pathogens of future pandemic potential.

Zinc oxide nanoparticles have garnered tremendous interest in bio and nanomedical applications and more so in cancer research. In the present work, zinc oxide nanoparticles were synthesized using the solution combustion method and characterized by UV-vis spectroscopy, FTIR, XRD, SEM and EDX. The infrared spectrum of the synthesized nanoparticles had a peak at 547 cm-1 which indicated the elongation frequency of the zinc bond to oxygen. The XRD spectrum was confirmed with the ICDD Data Card No: 36-1451 for zinc oxide and the average size of zinc oxide nanoparticles was estimated to be 17.51 nm. Scanning electron microscopy confirmed the presence of zinc oxide nanoparticles as Zincite with a hexagonal structure having an average diameter of 30.95 nm. The DNA binding interactions of the nanoparticles were studied using the absorption titration method. The nanoparticles could bind via the non-intercalation mode with a binding efficiency of 3.563x105 M-1. The synthesized zinc oxide nanoparticles exhibited cytotoxic activity against the mammalian cancer cell lines tested with the calculated IC50 values being 3.66 mu gmL-1 for MCF-7 (Breast), 2.91 mu gmL-1 for LN-18 (Glioblastoma-Brain), 23.14 mu gmL-1 for A-549 (Lung) and 94.33 mu gmL-1 for SHSY-5Y (Neuroblastoma-Bone). The highest cell death was observed for LN-18 glioblastoma cells of the brain. The study demonstrated a simple and conventional method of synthesis of zinc oxide nanoparticles that have demonstrated anticancer activity as evidenced by the in vitro cell viability assays. The outcome of this study could be applied to develop zinc oxide nanoparticles as anticancer agents with further validation in vivo.

Type 2 diabetes is currently treated by multiple drugs that are often combined to achieve maximum blood glucose lowering in patients. Yet more than 50% of patients are unable to attain target glucose levels. There is clear need for agents which can be added to current drugs to help patients achieve their target blood glucose levels safely. To this end we tested an anti-diabetic nutraceutical for its ability to enhance glucose uptake in muscle cells in combination with currently used cardiometabolic drugs. We show here that the nutraceutical is able to effectively improve glucose uptake of multiple drugs suggesting that it may help in enhancement of glucose lowering by the drugs in patients and achieve optimal glucose levels.

Summary Metformin is a widely used and is a safe anti-diabetic drug. It has also been shown to have anti-inflammatory and anti-viral activities in humans and animal models. Specifically we explored its activity in SARS-CoV-2 initiated COVID19 disease. Here we show that metformin 1. blocks the binding of SARS-CoV-2 spike protein receptor binding domain RBD to human ACE2 receptor 2. We also show that it has anti-inflammatory effects and reduces cytokine secretion as well as blocks the recruitment of monocytes to endothelial cells 3. Finally we show its activity in a hamster in vivo model of SARS-CoV-2 infection as a nasal formulation. Based on the safety and the therapeutic properties relevant to COVID-19 it is feasible to propose a nasal spray of metformin that can be used in treatment of this disease. A nasal spray would deliver the drug to the target organ lung and spare other organs which get exposed upon oral dosing.

There are many paths for the transmission of SarsCov2 virus. The main routes are nasal and oral and the droplets carrying the virus can also be transmitted thru ocular and skin tissues. The gastrointestinal (small intestine), nasal, ocular and skin tissues all present an acidic pH milieu and therefore any treatment with antibodies thru these routes has to have the antibodies remain active at acid pH as well as be resistant to typical protease digestion. To this end we profiled our anti-SarasCov2 receptor binding domain IgY antibodies for retention of activity at acidic and basic pHs and trypsin digestion. We find that the IgY are strongly resistant to denaturation at acid pH as well as not digested by trypsin. Our data strongly support the use of these IgY in treatment of viral transmission thru the GI, nasal, ocular and skin all tissues where the pH is acidic. We also provide an enabling platform to rapidly asses the suitability of any antibody or protein therapeutic for use at acidic pH.

Summary Despite the use of several drugs available to treat type 2 diabetes, many patients are unable to reach their target fasting plasma glucose and HbA1c levels. SGLT1 is the major intestinal transport transmembrane protein which functions in uptake of dietary glucose. If we antagonise the binding of dietary glucose to this transport protein, it is expected that blood glucose lowering will follow. We designed specific inhibitory avian antibodies (IgY) against the extracellular glucose binding domain of SGLT1 and tested their potential in glucose lowering. We demonstrate here the antibodies block uptake of glucose and improve the glycemic profile in vivo and represent a novel approach to inhibiting dietary glucose absorption as treatment for diabetes.

ABSTRACTPre diabetes and type 2 diabetes are increasingly becoming rampant world wide. While there are medications to control blood glucose in type 2 diabetics, currently there are no interventions prescribed for pre diabetes. Alternate strategies to control blood glucose are needed either to act alone in pre diabetes or as supplement to the existing drugs for type 2 diabetes. We report here the targeting of critical molecular steps in muscle glucose uptake and metabolism to result in glucose lowering using a combination of safe vitamins. Our in vitro and in vivo data support the potential for using such vitamin combination for glucose control in pre diabetes and as a supplement in type 2 diabetics.

Discovery and development of new drugs is a long, expensive and high risk proposition. Millions of dollars spent and decade plus years of time taken to discover a new drug have haunted pharma industry for many years. In part, the reliance on animal models to make go or no go decisions for selecting drugs for human trials has been a problem because animal biology does not capture human disease in entirety. In recognition of this, the last decade has seen the emergence of more human like tools being developed in the hope of better prediction of human outcomes. Towards that end we have developed a 3D bioprinted disease in a dish lung cancer model which uses human cells and includes ability to measure drug efficacy, toxicity and metabolism simultaneously. For drug profiling studies in our disease in a dish model we 3D bioprinted intestinal cells, layered below which were liver cells and finally underneath were target lung cancer cells. The idea was to simulate the path taken by an oral drug which encounters the gut, followed by liver and target organs. We demonstrate here that a 3D bioprinted disease model composed of human derived cells is able to concurrently measure in vitro drug efficacy, toxicity and metabolism. Such humanized models will help make early go or no go decisions on the potential of a drug to enter human trials.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Non caloric sweeteners (NCS) have been used for decades now as sugar substitutes in foods and beverages. The market for such products has grown immensely over time. There are human studies which report their negative effects on glucose metabolism with various results of disturbances in glucose metabolism, weight gain etc. No studies to the best of our knowledge have directly addressed the impact of the NCS on muscle glucose uptake. Muscle tissue can account for over 70 percent of whole-body glucose uptake. Therefore, we examined directly the effect of NCS on muscle cell glucose uptake. We find that aspartame moderately increased insulin stimulated glucose uptake by muscle cells in vitro. But sucralose, saccharin and stevia suppressed insulin stimulated glucose uptake. Sucralose is one of the most often used sweetener in foods and beverages globally it is important to understand its effects on glucose metabolism. Therefore, we explored the mechanism of its inhibition of glucose uptake by using an anti-diabetic nutraceutical which is known to target insulin mediated glucose uptake and metabolism pathways. We show here that the nutraceutical is able to relieve the suppression by sucrose in muscle uptake by a novel mechanism of action. We propose that such nutraceuticals may be useful to combine with sucralose containing products to offset negative effects of the NCS on glucose metabolism.

Novel drug discovery is a highly risky business where only 1 in 10,000–20,000 molecules successfully reaches the market. These poor success rates of new drug discovery have been attributed to poor translation of animal models to clinical outcomes. Regulatory agencies still do mandate extensive animal testing before and after a molecule enters clinical trials. Extensive efforts were made in the past three decades and revolutionary advances in (a) target-based, mechanistic models of drug discovery; (b) analytical techniques to detect the candidate drug at picomolar levels in the body fluids; (c) increased understanding of human systems biology pathways with omics platforms and other cutting-edge technologies; (d) cryopreserved banks of human primary cells; (e) digital databases that can retrieve required public information in minutes; (f) improved drug delivery technologies; (g) personalized and precision medicine tools in stratifying patient populations for clinical trial recruitments; and many more. Customized in vitro 3D models for specific clinical issues have been developed; however, they do not address the regulatory considerations to replace animals. 3D bioprinting, spheroids, and organoids of human tissues combined with microfluidics are becoming available and considerable progress has been made to mimic human physiology and drug disposition mechanisms. This review critically reviews the current state of the art of these in vitro 3D models.

The polyphenol E- and Z-gugggulsterone (GS) is an antagonist ligand for the Farnesoid X Receptor (FXR) and known to possess potent hypolipidemic properties as shown in various preclinical and clinical studies. In the present study, we examined drug-like properties of GS by assessing the isomers plasma protein binding, metabolic stability, CYP profiling, CYP inhibition, and phase I and II metabolite identification of GS using liver microsomes and S9 fractions. GS followed Lipinski and Veber rules and were substrates of CYP3A CYP2C19 and CYP2D6 isoforms. GS was also found to be an inhibitor of CYP2C19 with an IC50 value of 2.1 mu M. GS showed high plasma protein binding (<96%), and low to moderate binding with human serum albumin (similar to 70%). Unbound intrinsic clearances (CLint, (in-vitro)) was determined to be low at 0.029 +/- 0.0009 and 0.027 +/- 0.008 mL/min/mg protein for E- and Z-isomer, respectively in human liver microsomes. Nineteen phase I and II metabolites were identified and hydroxylation was found to be major metabolic pathway using human liver microsomes and S9 fractions. The results of in-vitro drug metabolism studies provide impetus for further structural modification of this pharmacophore in order to improve the stability of drugs with potent hypolipidemic effects. (C) 2018 Published by Elsevier B.V.

Modern drug discovery efforts have had mediocre success rates with increasing developmental costs, and this has encouraged pharmaceutical scientists to seek innovative approaches. Recently with the rise of the fields of systems biology and metabolomics, network pharmacology (NP) has begun to emerge as a new paradigm in drug discovery, with a focus on multiple targets and drug combinations for treating disease. Studies on the benefits of drug combinations lay the groundwork for a renewed focus on natural products in drug discovery. Natural products consist of a multitude of constituents that can act on a variety of targets in the body to induce pharmacodynamic responses that may together culminate in an additive or synergistic therapeutic effect. Although natural products cannot be patented, they can be used as starting points in the discovery of potent combination therapeutics. The optimal mix of bioactive ingredients in natural products can be determined via phenotypic screening. The targets and molecular mechanisms of action of these active ingredients can then be determined using chemical proteomics, and by implementing a reverse pharmacokinetics approach. This review article provides evidence supporting the potential benefits of natural product-based combination drugs, and summarizes drug discovery methods that can be applied to this class of drugs.

Anticancer efficacy of ginger phenolics (GPs) has been demonstrated in various in vitro assays and xenograft mouse models. However, only sub-therapeutic plasma concentrations of GPs were detected in human and mouse pharmacokinetic (PK) studies. Intriguingly, a significant portion of GPs occurred as phase II metabolites (mainly glucuronide conjugates) in plasma. To evaluate the disposition of GPs and understand the real players responsible for efficacy, we performed a PK and tissue distribution study in mice. Plasma exposure of GPs was similar on day 1 and 7, suggesting no induction or inhibition of clearance pathways. Both free and conjugated GPs accumulated in all tissues including tumors. While non-cytotoxicity of 6-ginerol glucuronide precluded the role of conjugated GPs in cell death, the free forms were cytotoxic against prostate cancer cells. The efficacy of ginger was best explained by the reconversion of conjugated GPs to free forms by β-glucuronidase, which is over-expressed in the tumor tissue. This previously unrecognized two-step process suggests an instantaneous conversion of ingested free GPs into conjugated forms, followed by their subsequent absorption into systemic circulation and reconversion into free forms. This proposed model uncovers the mechanistic underpinnings of ginger's anticancer activity despite sub-therapeutic levels of free GPs in the plasma.

Cytochrome P450 inhibition potential of Liv.52, snuff and camphor was assessed using human liver microsomes for any clinical consequences if taken together with other medications. They were screened for potential to inhibit 9 drug metabolizing cytochrome P450 (CYP 450) isoforms. Samples were analyzed by liquid chromatography mass spectrometry (LC-MS/MS) using stable labeled internal standards of metabolites. Liv.52 did not inhibit CYP2A6, CYP2D6, and CYP2E1 up to the highest tested concentration of 1.13 mg/mL. Its IC50 value ranged from 0.08 mg/mL to 0.160 mg/mL with CYP1A2, CYP2B6, CYP2C8, and CYP2C19. With CYP2C9, the IC50 value was 0.32 mg/mL, with CYP3A4 using midazolam as substrate, the mean IC50 value was 0.63 mg/mL and 0.86 mg/mL with testosterone as substrate. Snuff did not inhibit any of the tested CYPs up to the highest tested concentration of 500 μg/mL, except CYP2B6 and CYP2C8 with mean IC50 values of 381 μg/mL and 399 μg/mL, respectively. Camphor did not inhibit CYP1A2, CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 up to the highest tested concentration of 100 μg/mL. Camphor inhibited CYP2A6 and CYP2C19 with mean IC50 values of 60 μg/mL and 74 μg/mL, respectively. It showed the most potent inhibition with CYP2B6 with mean IC50 of 3.2 μg/mL. Generally, test items with IC50 values below 0.5 μg/mL are considered as potent CYP inhibitors and liable for further drug interaction studies. Liv.52 showed the lowest IC50 of 80 μg/mL with various CYPs, snuff showed IC50 values greater than 350 μg/mL with all the tested CYPs confirming no major CYP interaction liabilities. As not many marketed drugs are substrates of CYP2B6, the liability of camphor as CYP2B6 inhibitor is limited.

BACKGROUND:Drug transporters function as gatekeepers and modulate drug access into body and various tissues. Thus, a thorough and precise understanding of transporter liability for compound uptake and efflux is critical during drug development.METHODS:In the present study, we assessed the apparent permeability (Papp) and compared efflux ratio of various compounds in stably transfected Madin-Darby Canine Kidney (MDCKII) cells overexpressing human P-gp (MDCKII-MDR1), human BCRP (MDCKII-BCRP), wild-type (MDCKII-WT), and Caco-2 cell monolayers.RESULTS:We observed that quinidine, a substrate for MDR1 transporter, showed efflux ratio (Papp B-A/ Papp A-B) of 838 in MDCKII-MDR1 cells which plummeted to 14 in presence of verapamil, a known inhibitor of MDR1. With MDCKII-WT cells, Papp of quinidine dropped from 2 to 1, in the presence of verapamil. Caco-2 cells showed a diminutive decrease in efflux ratio of quinidine from 2.5 to 1.6 by verapamil. Prazosin and dantrolene were evaluated in MDCKII-BCRP cells and were found to have 80-fold higher efflux ratio compared to MDCKII-WT cells. In Caco-2 cells, prazosin and dantrolene showed efflux ratio of 4 and 2, respectively. Rhodamine-123, a fluorogenic probe substrate of MDR1 showed an efflux ratio of 4 in Caco-2 cells and BCRP substrate estrone-3-sulphate showed an efflux ratio of 7. In presence of BCRP inhibitor fumitremorgin-c, the efflux ratio of estrone-3-sulfate dropped to 1 in Caco-2 cells.CONCLUSION:The very high efflux ratios of MDR1 and BCRP substrates in transfected MDCKII cells clearly demonstrate the potential usefulness of these models to provide more definitive data to evaluate the transporter involvement compared to Caco-2 or MDCKII-WT cells.

The answer to the above questions primarily lies in the complexity of biology. Targeted drug discovery has become the mainstream effort of R&D in the last two decades. However, due to redundancy of network pathways and inherent adverse pharmacology of many targets, it is proving difficult for discovering and developing new drug candidates using a single target. About 87% of phase III clinical trials fail due to either lack of efficacy (66%) or safety (21%). Between 2007 and 2010, out of 83 molecules that failed in phase III trials, oncology (28%) and CNS disorders (18%) topped the list of failures (2.3).

Phytochemical complexity of plant extracts may offer health-promoting benefits including chemotherapeutic and chemopreventive effects. Isolation of 'most-active fraction' or single constituents from whole extracts may not only compromise the therapeutic efficacy but also render toxicity, thus emphasizing the importance of preserving the natural composition of whole extracts. The leaves of Annona muricata, commonly known as Graviola, are known to be rich in flavonoids, isoquinoline alkaloids and annonaceous acetogenins. Here, we demonstrate phytochemical synergy among the constituents of Graviola leaf extract (GLE) compared to its flavonoid-enriched (FEF) and acetogenin-enriched (AEF) fractions. Comparative quantitation of flavonoids revealed enrichment of rutin (~7-fold) and quercetin-3-glucoside (Q-3-G, ~3-fold) in FEF compared to GLE. In vivo pharmacokinetics and in vitro absorption kinetics of flavonoids revealed enhanced bioavailability of rutin in FEF compared to GLE. However, GLE was more effective in inhibiting in vitro prostate cancer proliferation, viability and clonogenic capacity compared to FEF. Oral administration of 100mg/kg bw GLE showed ~1.2-fold higher tumor growth-inhibitory efficacy than FEF in human prostate tumor xenografts although the concentration of rutin and Q-3-G was more in FEF. Contrarily, AEF, despite its superior in vitro and in vivo efficacy, resulted in death of the mice due to toxicity. Our data indicate that despite lower absorption and bioavailability of rutin, maximum efficacy was achieved in the case of GLE, which also comprises of other phytochemical groups including acetogenins that make up its natural complex environment. Hence, our study emphasizes on evaluating the nature of interactions among Graviola leaf phytochemcials for developing favorable dose regimen for prostate cancer management to achieve optimal therapeutic benefits.

Noscapine (Nos), an antitussive benzylisoquinoline opium alkaloid, is a non-toxic tubulin-binding agent currently in Phase II clinical trials for cancer chemotherapy. While preclinical studies have established its tumor-inhibitory properties in various cancers, poor absorptivity and rapid first-pass metabolism producing several uncharacterized metabolites for efficacy, present an impediment in translating its efficacy in humans. Here we report novel formulations of Nos in combination with dietary agents like capsaicin (Cap), piperine (Pip), eugenol (Eu) and curcumin (Cur) known for modulating Phase I and II drug metabolizing enzymes. In vivo pharmacokinetic (PK), organ toxicity evaluation of combinations, microsomal stability and in vitro cytochrome P450 (CYP) inhibition effects of Nos, Cap and Pip using human liver microsomes were performed. Single-dose PK screening of combinations revealed that the relative exposure of Nos (2μgh/mL) was enhanced by 2-fold (4μgh/mL) by Cap and Pip and their plasma concentration–time profiles showed multiple peaking phenomena for Nos indicating enterohepatic recirculation or differential absorption from intestine. CYP inhibition studies confirmed that Nos, Cap and Pip are not potent CYP inhibitors (IC50>1μM). Repeated oral dosing of Nos, Nos+Cap and Nos+Pip showed lower exposure (Cmax and AUClast) of Nos on day 7 compared to day 1. Nos Cmax decreased from 3087ng/mL to 684ng/mL and AUClast from 1024ngh/mL to 508ngh/mL. In presence of Cap and Pip, the decrease in Cmax and AUClast of Nos was similar. This may be due to potential enzyme induction leading to rapid clearance of Nos as the trend was observed in Nos alone group also. The lack of effect on intrinsic clearance of Nos suggests that the potential drug biotransformation modulators employed in this study did not contribute toward increased exposure of Nos on repeated dosing. We envision that Nos-induced enzyme induction could alter the therapeutic efficacy of co-administered drugs, hence emphasizing the need for strategic evaluation of the metabolism of Nos to reap its maximum efficacy.

There are various factors which can affect the quality of PK data such as stress during animal handling [1], blood loss with serial sampling [2], feed [3,4], age and gender [5].Another important factor to consider is blood sampling site like retroorbital puncture [6], tail vein [6], Saphenous vein [6,7], jugular vein [8][9][10], sublingual [11], and tail snip [12], all of which have their own inherent advantages and disadvantages.According to published literature, in a day (24 h), no more than 10% of total Abstract Pharmacokinetic (PK) studies play an important role in identifying lead compounds for further development.Typically rats are used for PK screening of New Chemical Entities (NCEs) as the compound requirements are minimal (<10 mg), multiple blood sampling (up to 10 samples) can be performed from the same animal and in small volumes (5-25μL) for sample analysis.Blood sampling site is critical in obtaining multiple blood samples of good quality and in small volumes with minimal stress to animals.However, it is not known whether PK parameters can be influenced by sampling site.Thus, in this study, we evaluated the effect of different blood sampling sites like retro-orbital plexus, jugular vein and saphenous vein on PK parameters of Dapsone.Dapsone was administered both orally and intravenously at a dose of 12 mg/kg to a group of 4 male Sprague Dawley rats and blood samples were collected up to 24 h.Samples were analyzed by LC/MS/MS and PK parameters were calculated.With all the sampling techniques, PK parameters like clearance, volume of distribution, half-life and bioavailability were similar.Due to the control on the blood volume withdrawn at each time point, quick sampling with minimal hemolysis and minimal animal handling stress during sampling, Jugular Vein (JV) or Saphenous Vein (SV) sampled rats can be used for PK studies.Further for saphenous sampling no pre-study preparation like cannulation is required before dosing the animals therefore sampling of rats through saphenous vein is recommended for pharmacokinetic and toxicokinetic studies.To summarize, SV sampling reduce the number of animals in different Pharmacokinetic (PK) (mouse) and Toxicokinetic (TK) (mouse and rat) studies by using serial draws, offers reduction and refinement over the othersampling techniques with minimal preparation upfront and with a potential to replace them.