Search results for: pharmacodynamics

Authors: Yong Cang

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RJV001 is a subcutaneous injection containing mutated recombinant Collagenase H (ColH), leading to disruption of collagen matrix in adipose tissue and programmed cell death of adipocytes. Here we reported our clinical investigation of the safety, tolerance and pharmacodynamics of localized RJV001 injection into healthy human abdominal fat tissues (NCT04821648, Arizona Research Center). Investigate the safety, tolerance and clinical pharmacodynamics of subcutaneous RJV001 in humans. In the dose-escalating study, 18 subjects completed the study, 100% female, 78% white, with a mean age of 42[±9.9]. All three tested dose (0.05, 0.075 and 0.15 mg/injection), up to 30 injections, were safe and well-tolerated. Bruising and tenderness to palpation, mild to moderate, were the most frequent local skin reactions but nearly all resolved within 30 days. Additionally, physician-monitored ultrasound measurement showed that a reduction in abdominal fat tissue thickness was consistently observed in Cohort C (0.075, 0.15 mg/injection, 30injections), with a mean reduction of 7.37 [± 2.020] mm. Based on this clinical study, RJV001 has been advanced to phase II clinical studies. In the dose-escalating study, subcutaneously administered RJV001 was safe and well-tolerated in healthy adults up to 0.15 mg/injection, 30 injections. Fat reduction and adipocytolysis were observed by ultrasound measurements and histological analysis for exploratory purposes.

Keywords: fat reduction, mutant collagenase, clinical trial, subcutaneous injection

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Authors: Malay K. Das, Bhanu P. Sahu

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Furosemide is a weakly acidic diuretic indicated for treatment of edema and hypertension. It has very poor solubility but high permeability through stomach and upper gastrointestinal tract (GIT). Due to its limited solubility it has poor and variable oral bioavailability of 10-90%. The aim of this study was to enhance the oral bioavailability of furosemide by preparation of nanosuspensions. The nanosuspensions were prepared by nanoprecipitation with sonication using DMSO (dimethyl sulfoxide) as a solvent and water as an antisolvent (NA). The prepared nanosuspensions were sterically stabilized with polyvinyl acetate (PVA).These were characterized for particle size, ζ potential, polydispersity index, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD) pattern and release behavior. The effect of nanoprecipitation on oral bioavailability of furosemide nanosuspension was studied by in vitro dissolution and in vivo absorption study in rats and compared to pure drug. The stable nanosuspension was obtained with average size range of the precipitated nanoparticles between 150-300 nm and was found to be homogenous showing a narrow polydispersity index of 0.3±0.1. DSC and XRD studies indicated that the crystalline furosemide drug was converted to amorphous form upon precipitation into nanoparticles. The release profiles of nanosuspension formulation showed up to 81.2% release in 4 h. The in vivo studies on rats revealed a significant increase in the oral absorption of furosemide in the nanosuspension compared to pure drug. The AUC0→24 and Cmax values of nanosuspension were approximately 1.38 and 1.68-fold greater than that of pure drug, respectively. Furosemide nanosuspension showed 20.06±0.02 % decrease in systolic blood pressure compared to 13.37±0.02 % in plain furosemide suspension, respectively. The improved oral bioavailability and pharmacodynamics effect of furosemide may be due to the improved dissolution of furosemide in simulated gastric fluid which results in enhanced oral systemic absorption of furosemide from stomach region where it has better permeability.

Keywords: furosemide, nanosuspension, bioavailability enhancement, nanoprecipitation, oral drug delivery

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Authors: Sheraz Gul

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The requirements for progressing a compound to clinical trials is well established and relies on the results from in-vitro and in-vivo animal tests to indicate that it is likely to be safe and efficacious when testing in humans. The typical data package required will include demonstrating compound safety, toxicity, bioavailability, pharmacodynamics (potential effects of the compound on body systems) and pharmacokinetics (how the compound is potentially absorbed, distributed, metabolised and eliminated after dosing in humans). If the desired criteria are met and the compound meets the clinical Candidate criteria and is deemed worthy of further development, a submission to regulatory bodies such as the US Food & Drug Administration for an exploratory Investigational New Drug Study can be made. The purpose of this study is to collect data to establish that the compound will not expose humans to unreasonable risks when used in limited, early-stage clinical studies in patients or normal volunteer subjects (Phase I). These studies are also designed to determine the metabolism and pharmacologic actions of the drug in humans, the side effects associated with increasing doses, and, if possible, to gain early evidence on their effectiveness. In order to reach the above goals, we have developed a pre-clinical high throughput Absorption, Distribution, Metabolism and Excretion–Toxicity (ADME–Toxicity) panel of assays to identify compounds that are likely to meet the Lead and Candidate compound acceptance criteria. This panel includes solubility studies in a range of biological fluids, cell viability studies in cancer and primary cell-lines, mitochondrial toxicity, off-target effects (across the kinase, protease, histone deacetylase, phosphodiesterase and GPCR protein families), CYP450 inhibition (5 different CYP450 enzymes), CYP450 induction, cardio-toxicity (hERG) and gene-toxicity. This panel of assays has been applied to multiple compound series developed in a number of projects delivering Lead and clinical Candidates and examples from these will be presented.

Keywords: absorption, distribution, metabolism and excretion–toxicity , drug discovery, food and drug administration , pharmacodynamics

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Authors: Alya A. Arabi

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Bioisosteres are functional groups that can be interchangeably used without affecting the potency of the drug. Bioisosteres have similar pharmacological properties. Bioisosterism is useful for modifying the physicochemical properties of a drug while obeying the Lipinski’s rules. Bioisosteres are key in optimizing the pharmacokinetic and pharmacodynamics properties of a drug. Tetrazole and carboxylate anions are non-classic bioisosteres. Density functional theory was used to obtain the wavefunction of the molecules and the optimized geometries. The quantum theory of atoms in molecules (QTAIM) was used to uncover the similarity of the average electron density in tetrazole and carboxylate anions. This similarity between the bioisosteres capped by a methyl group was valid despite the fact that the groups have different volumes, charges, energies, or electron populations. The biochemical correspondence of tetrazole and carboxylic acid was also determined to be a result of the similarity of the topography of the electrostatic potential (ESP). The ESP demonstrates the pharmacological and biochemical resemblance for a matching “key-and-lock” interaction.

Keywords: bioisosteres, carboxylic acid, density functional theory, electrostatic potential, tetrazole

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Authors: A. Mech

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Repetitive transcranial magnetic stimulation has proven to be a valuable treatment option for patients who have failed to respond to multiple courses of antidepressant medication. In fact, the American Psychiatric Association recommends TMS after one failed treatment course of antidepressant medication. Genetic testing has proven valuable for pharmacokinetic variables, which, if understood, could lead to more efficient dosing of psychotropic medications to improve outcomes. Pharmacodynamic testing can identify biomarkers, which, if addressed, can improve patients' outcomes in antidepressant therapy. Monotherapy treatment of major depressive disorder with methylated B vitamin treatment has been shown to be safe and effective in patients with MTHFR polymorphisms without waiting for multiple trials of failed medication treatment for depression. Such treatment has demonstrated remission rates similar to antidepressant clinical trials. Combining pharmacodynamics testing with repetitive TMS treatment with NeuroStar has shown promising potential for enhancing remission rates and durability of treatment. In this study, a retrospective chart review (ongoing) of patients who obtained repetitive TMS treatment enhanced by dietary supplementation guided by Pharmacodynamic testing, displayed a greater remission rate (90%) than patients treated with only NeuroStar TMS (62%).

Keywords: improved remission rate, major depressive disorder, pharmacodynamic testing, rTMS outcomes

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Authors: Ehab El-Dabaa, Omnia Ali, Mohamed Abd El-Hady, Ahmed Osman

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Recombinant human interferon alpha 2 (rhIFN-α2) is FDA approved for treatment of some viral and malignant diseases. Approved pegylated rhIFN-α2 drugs have highly improved pharmacokinetics, pharmacodynamics and therapeutic efficiency compared to native protein. In this work, we studied the pegylation of purified properly refolded rhIFN-α2b using linear 20kDa PEG-NHS (polyethylene glycol- N-hydroxysuccinimidyl ester) to prepare pegylated rhIFN-α2b with high stability and activity. The effect of different parameters like rhIFN-α2b final concentration, pH, rhIFN-α2b/PEG molar ratios and reaction time on the efficiency of pegylation (high percentage of monopegylated rhIFN-α2b) have been studied in small scale (100µl) pegylation reaction trials. Study of the percentages of different components of these reactions (mono, di, polypegylated rhIFN-α2b and unpegylated rhIFN-α2b) indicated that 2h is optimum time to complete the reaction. The pegylation efficiency increased at pH 8 (57.9%) by reducing the protein concentration to 1mg/ml and reducing the rhIFN-α2b/PEG ratio to 1:2. Using larger scale pegylation reaction (65% pegylation efficiency), ion exchange chromatography method has been optimized to prepare and purify the monopegylated rhIFN-α2b with high purity (96%). The prepared monopegylated rhIFN-α2b had apparent Mwt of approximately 65 kDa and high in vitro antiviral activity (2.1x10⁷ ± 0.8 x10⁷ IU/mg). Although it retained approximately 8.4 % of the antiviral activity of the unpegylated rhIFN-α2b, its activity is high compared to other pegylated rhIFN-α2 developed by using similar approach or higher molecular weight branched PEG.

Keywords: antiviral activity, rhIFN-α2b, pegylation, pegylation efficiency

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Authors: Murali Munisamy, Gauthaman Karunakaran, Mubarak Al-Gahtany, Vivekanandhan Subbiah, M. Manjari Tripati

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Background: Sodium valproate is a widely prescribed broad-spectrum anti-epileptic drug. It shows high inter-individual variability in pharmacokinetics and pharmacodynamics and has a narrow therapeutic range. We evaluated the effects of polymorphic uridine diphosphate glucuronosyltransferase (UGT1A9) metabolizing enzyme on the pharmacokinetics of sodium valproate in the patients with epilepsy who showed toxicity to therapy. Methods: Genotype analysis of the patients was made with polymerase chain–restriction fragment length polymorphism (RFLP) with sequencing. Plasma drug concentrations were measured with reversed phase high-performance liquid chromatography (HPLC) and concentration–time data were analyzed by using a non-compartmental approach. Results: The results of this study suggested a significant genotypic as well as allelic association with valproic acid toxicity for UGT1A9 polymorphic enzymes. The elimination half-life (t 1/2=40.2 h) of valproic acid was longer and the clearance rate (CL=937 ml/h) was lower in the poor metabolizers group of UGT1A9 polymorphism who showed toxicity than in the intermediate metabolizers group (t1/2=35.5 h, CL=1042 ml/h) or the extensive metabolizers group (t1/2=26. h, CL=1,302 ml/h). Conclusion: Our findings suggest that the UGT1A9 genetic polymorphism plays a significant role in the steady state concentration of sodium valproate, and it thereby has an impact on the toxicity of the sodium valproate used in the patients with epilepsy.

Keywords: UGT1A9, sodium valporate, pharmacogenetics, polymorphism

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Authors: Ruqaiya Khalil, Saman Usmani, Zaheer Ul-Haq

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The accurate characterization of ligand binding affinity is indispensable for designing molecules with optimized binding affinity. Computational tools help in many directions to predict quantitative correlations between protein-ligand structure and their binding affinities. Molecular dynamics (MD) simulation is a modern state-of-the-art technique to evaluate the underlying basis of ligand-protein interactions by characterizing dynamic and energetic properties during the event. Autoimmune diseases arise from an abnormal immune response of the body against own tissues. The current regimen for the described condition is limited to immune-modulators having compromised pharmacodynamics and pharmacokinetics profiles. One of the key player mediating immunity and tolerance, thus invoking autoimmunity is Interleukin-2; a cytokine influencing the growth of T cells. Molecular dynamics simulation techniques are applied to seek insight into the inhibitory mechanisms of newly synthesized compounds that manifested immunosuppressant potentials during in silico pipeline. In addition to estimation of free energies associated with ligand binding, MD simulation yielded us a great deal of information about ligand-macromolecule interactions to evaluate the pattern of interactions and the molecular basis of inhibition. The present study is a continuum of our efforts to identify interleukin-2 inhibitors of both natural and synthetic origin. Herein, we report molecular dynamics simulation studies of Interluekin-2 complexed with different antagonists previously reported by our group. The study of protein-ligand dynamics enabled us to gain a better understanding of the contribution of different active site residues in ligand binding. The results of the study will be used as the guide to rationalize the fragment based synthesis of drug-like interleukin-2 inhibitors as immune-modulators.

Keywords: immuno-modulators, MD simulation, protein-ligand interaction, structure-based drug design

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Authors: L. L. Tundisi, A. Pessoa Jr., E. B. Tambourgi, E. Silveira, P. G. Mazzola

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L-asparaginase (L-ASNase) is the gold standard treatment for acute lymphoblastic leukemia that mainly affects pediatric patients; treatment increases survival from 20% to 90%. The characterization of other L-Asparaginases, apart from the most used from Escherichia coli and Erwinia chrysanthemi, has been reported, but the choice of the most appropriate is still under debate. This choice should be based on its pharmacokinetics, immune hypersensitivity, doses, prices, pharmacodynamics. The main factors influencing the antileukemic activity of ASNase are enzymatic activity, Km, glutaminase activity, clearance of the enzyme and development of resistance. However, most of the commercialized enzyme present an intrinsic glutaminase activity, which is responsible for some side effects. In this study, glutaminase free asparaginase produced from Aspergillus oryzae was precipitated in different percentages of ethanol (0–80%), until optimum ethanol concentration of 60% (w/w) was found. Following, precipitation of crude L-ASNase was performed in a single step, using 60% (w/w) ethanol, under constant agitation and temperature. It presented activity of 135.45 U/mg and after gel filtration chromatography with Sephadex G-the enzymatic activity was 322.02 U/mg. The apparent molecular mass of the purified L-ASNase fraction was estimated by 10% SDS-PAGE. Proteins were stained with Coomassie Brilliant Blue R-250. The molar mass range was from 10 kDa to 250 kDa. L-ASNase from Aspergillus oryzae was characterized aiming possible therapeutic use. Four different buffers (phosphate-citrate buffer pH 2.6 to 5.8; phosphate buffer pH 5.8 to 7.4; Tris - HCl pH 7.4 to 9.0; and carbonate buffer pH 9.8 to 10.6) were used to measure the optimum pH for L-ASNase activity. The optimum temperature for enzyme activity was measured at optimal pH conditions (Tris-HCl and phosphate buffer, pH 7.4) at different temperatures ranging from 5 to 55°C. All activities were calculated by quantifying the free ammonia, using the Nessler reagent. The kinetic parameters calculation, e.g. Michaelis-Menten constant (Km), maximum velocity (Vmax) and Hills coefficient (n), were performed by incubating the enzyme in different concentrations of the substrate at optimum conditions of pH and fitted on Hill’s equation. This glutaminase free asparaginase showed a low Km (3.39 mM and 3.81 mM) and enzymatic activity of 135.45 U/mg after precipitation with ethanol. After gel filtration chromatography it rose to 322.02 U/mg. Optimum activity was found between pH 5.8 - 9.0, best activity results with phosphate buffer pH 7.4 and Tris-HCl pH 7.4 and showed activity from 5°C to 55°C. These results indicate that L-ASNase from A. oryzae has the potential for human use.

Keywords: biopharmaceuticals, bioprocessing, bioproducts, biotechnology, enzyme activity, ethanol precipitation

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Authors: Md. Fazlul Karim, Ashik Sharfaraz, Aysha Ferdoushi

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Background: Computational medicinal chemistry approaches are used for designing and identifying new drug-like molecules, predicting properties and pharmacological activities, and optimizing lead compounds in drug development. SIRT7, a nicotinamide adenine dinucleotide (NAD+)-dependent deacylase which regulates aging, is an emerging target for cancer therapy with mounting evidence that SIRT7 downregulation plays important roles in reversing cancer phenotypes and suppressing tumor growth. Activation or altered expression of SIRT7 is associated with the progression and invasion of various cancers, including liver, breast, gastric, prostate, and non-small cell lung cancer. Objectives: The goal of this work was to identify potent and selective bioactive candidate inhibitors of SIRT7 by in silico screening of small molecule compounds obtained from Nigella sativa (N. sativa). Methods: SIRT7 structure was retrieved from The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), and its active site was identified using CASTp and metaPocket. Molecular docking simulation was performed with PyRx 0.8 virtual screening software. Drug-likeness properties were tested using SwissADME and pkCSM. In silico toxicity was evaluated by Osiris Property Explorer. Bioactivity was predicted by Molinspiration software. Antitumor activity was screened for Prediction of Activity Spectra for Substances (PASS) using Way2Drug web server. Molecular dynamics (MD) simulation was carried out by Desmond v3.6 package. Results: A total of 159 bioactive compounds from the N. Sativa were screened against the SIRT7 enzyme. Five bioactive compounds: chrysin (CID:5281607), pinocembrin (CID:68071), nigellidine (CID:136828302), nigellicine (CID:11402337), and epicatechin (CID:72276) were identified as potent SIRT7 anti-cancer candidates after docking score evaluation and applying Lipinski's Rule of Five. Finally, MD simulation identified Chrysin as the top SIRT7 anti-cancer candidate molecule. Conclusion: Chrysin, which shows a potential inhibitory effect against SIRT7, can act as a possible anti-cancer drug candidate. This inhibitor warrants further evaluation to check its pharmacokinetics and pharmacodynamics properties both in vitro and in vivo.

Keywords: SIRT7, antitumor, molecular docking, molecular dynamics simulation

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Authors: Farah Diab, Mohamad Khalil, Francesca Storace, Francesca Baldini, Piero Portincasaa, Giulio Lupidi, Laura Vergani

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Thymbra spicata is a thyme-like plant belonging to the Lamiaceae family that shows a global distribution, especially in the eastern Mediterranean region. Leaves of T. spicata contain large amounts of phenols such as phenolic acids (rosmarinic acid), phenolic monoterpenes (carvacrol), and flavonoids. In Lebanon, T. spicata is currently used as a culinary herb in salad and infusion, as well as for traditional medicinal purposes. Carvacrol (5-isopropyl-2-methyl phenol), the most abundant polyphenol in the organic extract and essential oils, has a great array of pharmacological properties. In fact, carvacrol is largely employed as a food additive and neutraceutical agent. Our aim is to investigate the beneficial effects of T. spicata ethanolic extract (TE) and its main component, carvacrol, using in vitro models of hepatic steatosis and endothelial dysfunction. As a further point, we focused on investigating if and how the binding of carvacrol to albumin, the physiological transporter for drugs in the blood, might be altered by the presence of high levels of fatty acids (FAs), thus impairing the carvacrol bio-distribution in vivo. For that reason, hepatic FaO cells treated with exogenous FAs such as oleate and palmitate mimic hepatosteatosis; endothelial HECV cells exposed to hydrogen peroxide are a model of endothelial dysfunction. In these models, we measured lipid accumulation, free radical production, lipoperoxidation, and nitric oxide release before and after treatment with carvacrol. The carvacrol binding to albumin with/without high levels of long-chain FAs was assessed by absorption and emission spectroscopies. Our findings show that both TE and carvacrol (i) counteracted lipid accumulation in hepatocytes by decreasing the intracellular and extracellular lipid contents in steatotic FaO cells; (ii) decreased oxidative stress in endothelial cells by significantly reducing lipoperoxidation and free radical production, as well as, attenuating the nitric oxide release; (ii) high levels of circulating FAs reduced the binding of carvacrol to albumin. The beneficial effects of TE and carvacrol on both hepatic and endothelial cells point to a nutraceutical potential. However, high levels of circulating FAs, such as those occurring in metabolic disorders, might hinder the carvacrol transport, bio-distribution, and pharmacodynamics.

Keywords: carvacrol, endothelial dysfunction, fatty acids, non-alcoholic fatty liver diseases, serum albumin

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Authors: Mumuni Sumaila, Viness Pillay, Yahya E. Choonara, Pradeep Kumar, Pierre P. Kondiah

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Tuberculosis (TB) remains a serious challenge to public health globally, despite every effort put together to curb the disease. Current TB therapeutics available have proven to be inefficient due to a multitude of drawbacks that range from serious adverse effects/drug toxicity to inconsistent bioavailability, which ultimately contributes to the emergence of drug-resistant TB. An effective ‘cargo’ system designed to cleverly deliver therapeutic doses of anti-TB drugs to infection sites and in a sustained-release manner may provide a better therapeutic choice towards winning the war against TB. In the current study, we investigated mannose-functionalized lipopolysaccharide hybrid nanoparticles for safety and efficacy towards macrophage-targeted simultaneous delivery of the two first-line anti-TB drugs, rifampicin (RF) and isoniazid (IS). RF-IS-loaded lipopolysaccharide hybrid nanoparticles were fabricated using the solvent injection technique (SIT), incorporating soy lecithin (SL) and low molecular weight chitosan (CS) as the lipid and polysaccharide components, respectively. Surface-functionalized nanoparticles were obtained through the reaction of the aldehyde group of mannose with free amine functionality present at the surface of the nanoparticles. The functionalized nanocarriers were spherical with average particle size and surface charge of 107.83 nm and +21.77 mV, respectively, and entrapment efficiencies (EE) were 53.52% and 69.80% for RF and IS, respectively. FTIR spectrum revealed high-intensity bands between 1663 cm⁻¹ and 1408 cm⁻¹ wavenumbers (absent in non-functionalized nanoparticles), which could be attributed to the C=N stretching vibration produced by the formation of Schiff’s base (–N=CH–) during the mannosylation reaction. In vitro release studies showed a sustained-release profile for RF and IS, with less than half of the total payload released over a 48-hour period. The nanocarriers were biocompatible and safe, with more than 80% cell viability achieved when incubated with RAW 264.7 cells at concentrations 30 to 500 μg/mL over a 24-hour period. Cellular uptake studies (after a 24-hour incubation period with the murine macrophage cells, RAW 264.7) revealed a 13- and a 9-fold increase in intracellular accumulation of RF and IS, respectively, when compared with the unformulated RF+IS solution. A 6- and a 3-fold increase in intracellular accumulation of RF and IS, respectively, were observed when compared with the non-functionalized nanoparticles. Furthermore, fluorescent microscopy images showed nanoparticle internalization and accumulation within the RAW 264.7 cells, which was more significant in the mannose-functionalized system compared to the non-functionalized nanoparticles. The overall results suggested that the fabricated mannose-functionalized lipopolysaccharide nanoparticles are a safe and promising platform for macrophage-targeted delivery of anti-TB therapeutics. However, in vivo pharmacokinetic/pharmacodynamics studies are required to further substantiate the therapeutic efficacy of the nanosystem.

Keywords: anti-tuberculosis therapeutics, hybrid nanosystem, lipopolysaccharide nanoparticles, macrophage-targeted delivery

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Authors: Juliette Begin, Juliano Colapelle, Andrea Taratanu, Daniel Thirion, Amelie Marsot, Bryan A. Ross

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Chronic obstructive pulmonary disease (COPD), a leading cause of death globally, is characterized by chronic airflow obstruction and acute exacerbations (AECOPDs) that are often triggered by respiratory infections. Pseudomonas aeruginosa (P. aeruginosa), a potentially serious bacterial cause of AECOPDs, is treated with targeted anti-pseudomonal antibiotics. These select few antimicrobials are often used as first-line therapy in patients who are clinically unwell and/or in those suspected of P. aeruginosa-related infection prior to confirmation, potentially contributing to antimicrobial resistance. The present study evaluates prescribing practices in patients with a confirmed sputum history of P. aeruginosa admitted for AECOPD at the McGill University Health Centre (MUHC) and treated with anti-pseudomonal antibiotics. Serum antibiotic concentrations were measured from the same-day peak, trough, and mid-dose blood sampling intervals after reaching steady-state (on or after day 3) and were quantified using ultra-high-performance liquid chromatography (UHPLC). Demographic, clinical, and treatment outcomes were extracted from patient medical charts. Treatment failure was defined by respiratory-related death or mechanical ventilation after ≥3 days of antibiotics; antibiotic therapy extended beyond 2 weeks or a new antibiotic regimen started; or urgent care readmission within 30 days for AECOPD. To date, 9 of 30 planned participants have completed testing: seven received ciprofloxacin, one received meropenem, and one received piperacillin-tazobactam. Due to serum sample batching requirements, the serum ciprofloxacin concentration results for the first 2/8 participants are presented at the time of writing. The first participant had serum levels of 5.45mg/L (T₀), 4.74mg/L (T₅₀), and 4.49mg/L (T₁₀₀), while the second had serum levels of 5mg/L (T₀), 2.6mg/L (T₅₀), and 2.51mg/L (T₁₀₀). Pharmacokinetic parameters Cmax (5.18±0.43mg/L), T₁/₂ (23.56±18.94hours), and AUC (181.9±155.95mg*h/l) were higher than reported monograph values and met target AUC-to-MIC ratio of >125. The patients treated with meropenem and with piperacillin-tazobactam experienced treatment failure. Preliminary results suggest that standard ciprofloxacin dosing in patients experiencing an AECOPD and colonized with P. aeruginosa appears to achieve effective serum concentrations. Final cohort results will inform the pharmacokinetic appropriateness and clinical sufficiency of current AECOPD antimicrobial strategies in P. aeruginosa-colonized patients. This study will guide clinicians in determining the appropriate dosing for AECOPD treatment to achieve therapeutic levels, optimizing outcomes, and minimizing adverse effects. It could also highlight the value of routine antibiotic level monitoring in patients with treatment failure to ensure optimal serum concentrations.

Keywords: acute exacerbation, antimicrobial resistance, chronic obstructive pulmonary disease, pharmacokinetics/pharmacodynamics, Pseudomonas aeruginosa

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Authors: Shrestha Sharma, Jasjeet K. Sahni, Javed Ali, Sanjula Baboota

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Introduction: Rutin is a naturally occurring strong antioxidant molecule belonging to bioflavonoid category. Due to its free radical scavenging properties, it has been found to be beneficial in the treatment of various diseases including inflammation, cancer, diabetes, allergy, cardiovascular disorders and various types of microbial infections. Despite its beneficial effects, it suffers from the problem of low aqueous solubility which is responsible for low oral bioavailability. The aim of our study was to optimize and characterize self-nanoemulsifying drug delivery system (SNEDDS) of rutin using Box-Behnken design (BBD) combined with a desirability function. Further various antioxidant, pharmacokinetic and pharmacodynamic studies were performed for the optimized rutin SNEDDS formulation. Methodologies: Selection of oil, surfactant and co-surfactant was done on the basis of solubility/miscibility studies. Sefsol+ Vitamin E, Solutol HS 15 and Transcutol P were selected as oil phase, surfactant and co-surfactant respectively. Optimization of SNEDDS formulations was done by a three-factor, three-level (33)BBD. The independent factors were Sefsol+ Vitamin E, Solutol HS15, and Transcutol P. The dependent variables were globule size, self emulsification time (SEF), % transmittance and cumulative percentage drug released. Various response surface graphs and contour plots were constructed to understand the effect of different factor, their levels and combinations on the responses. The optimized Rutin SNEDDS formulation was characterized for various parameters such as globule size, zeta potential, viscosity, refractive index , % Transmittance and in vitro drug release. Ex vivo permeation studies and pharmacokinetic studies were performed for optimized formulation. Antioxidant activity was determined by DPPH and reducing power assays. Anti-inflammatory activity was determined by using carrageenan induced rat paw oedema method. Permeation of rutin across small intestine was assessed using confocal laser scanning microscopy (CLSM). Major findings:The optimized SNEDDS formulation consisting of Sefsol+ Vitamin E - Solutol HS15 -Transcutol HP at proportions of 25:35:17.5 (w/w) was prepared and a comparison of the predicted values and experimental values were found to be in close agreement. The globule size and PDI of optimized SNEDDS formulation was found to be 16.08 ± 0.02 nm and 0.124±0.01 respectively. Significant (p˂0.05) increase in percentage drug release was achieved in the case of optimized SNEDDS formulation (98.8 %) as compared to rutin suspension. Furthermore, pharmacokinetic study showed a 2.3-fold increase in relative oral bioavailability compared with that of the suspension. Antioxidant assay results indicated better efficacy of the developed formulation than the pure drug and it was found to be comparable with ascorbic acid. The results of anti-inflammatory studies showed 72.93 % inhibition for the SNEDDS formulation which was significantly higher than the drug suspension 46.56%. The results of CLSM indicated that the absorption of SNEDDS formulation was considerably higher than that from rutin suspension. Conclusion: Rutin SNEDDS have been successfully prepared and they can serve as an effective tool in enhancing oral bioavailability and efficacy of Rutin.

Keywords: rutin, oral bioavilability, pharamacokinetics, pharmacodynamics

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Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

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The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

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Authors: Amandeep Thakur, Yi-Hsuan Chu, Chun-Hsu Pan, Kunal Nepali

Abstract:

The transfer of ADP-ribose residues onto target substrates from nicotinamide adenine dinucleotide (NAD) (PARylation) is catalyzed by Poly (ADP-ribose) polymerases (PARPs). Amongst the PARP family members, the DNA damage response in cancer is majorly regulated by PARP1 and PARP2. The blockade of DNA repair by PARP inhibitors leads to the progression of DNA single-strand breaks (induced by some triggering factors) to double-strand breaks. Notably, PARP inhibitors are remarkably effective in cancers with defective homologous recombination repair (HRR). In particular, cancer cells with BRCA mutations are responsive to therapy with PARP inhibitors. The aforementioned requirement for PARP inhibitors to be effective confers a narrow activity spectrum to PARP inhibitors, which hinders their clinical applicability. Thus, the quest to expand the application horizons of PARP inhibitors beyond BRCA mutations is the need of the hour. Literature precedents reveal that HDAC inhibition induces BRCAness in cancer cells and can broaden the therapeutic scope of PARP inhibitors. Driven by such disclosures, dual inhibitors targeting both PARP and HDAC enzymes were designed by our research group to extend the efficacy of PARP inhibitors beyond BRCA-mutated cancers to cancers with induced BRCAness. The design strategy involved the installation of Veliparib, an investigational PARP inhibitor, as a surface recognition part in the HDAC inhibitor pharmacophore model. The chemical architecture of veliparib was deemed appropriate as a starting point for the generation of dual inhibitors by virtue of its size and structural flexibility. A validatory docking study was conducted at the outset to predict the binding mode of the designed dual modulatory chemical architectures. Subsequently, the designed chemical architectures were synthesized via a multistep synthetic route and evaluated for antitumor efficacy. Delightfully, one compound manifested impressive anti-leukemic effects (HL-60 cell lines) mediated via dual inhibition of PARP and class I HDACs. The outcome of the western blot analysis revealed that the compound could downregulate the expression levels of PARP1 and PARP2 and the HDAC isoforms (HDAC1, 2, and 3). Also, the dual PARP-HDAC inhibitor upregulated the protein expression of the acetyl histone H3, confirming its abrogation potential for class I HDACs. In addition, the dual modulator could arrest the cell cycle at the G0/G1 phase and induce autophagy. Further, polymer-based nanoformulation of the dual inhibitor was furnished to afford targeted delivery of the dual inhibitor at the cancer site. Transmission electron microscopy (TEM) results indicate that the nanoparticles were monodispersed and spherical. Moreover, the polymeric nanoformulation exhibited an appropriate particle size. Delightfully, pH-sensitive behavior was manifested by the polymeric nanoformulation that led to selective antitumor effects towards the HL-60 cell lines. In light of the magnificent anti-leukemic profile of the identified dual PARP-HDAC inhibitor, in-vivo studies (pharmacokinetics and pharmacodynamics) are currently being conducted. Notably, the optimistic findings of the aforementioned study have spurred our research group to initiate several medicinal chemistry campaigns to create bifunctional small molecule inhibitors addressing PARP as the primary target.

Keywords: PARP inhibitors, HDAC inhibitors, BRCA mutations, leukemia

Procedia PDF Downloads 23

Authors: Jerome Dalphinis, Vishal Patel

Abstract:

The Bailiwick of Jersey is an island British crown dependency situated off the coast of France. Jersey General Hospital’s emergency department sees approximately 40,000 patients a year. It’s outside the NHS, with secondary care being free at the point of care. Sedation is a continuum which extends from a normal conscious level to being fully unresponsive. Procedural sedation produces a minimally depressed level of consciousness in which the patient retains the ability to maintain an airway, and they respond appropriately to physical stimulation. The goals of it are to improve patient comfort and tolerance of the procedure and alleviate associated anxiety. Indications can be stratified by acuity, emergency (cardioversion for life-threatening dysrhythmia), and urgency (joint reduction). In the emergency department, this is most often achieved using a combination of opioids and benzodiazepines. Some departments also use ketamine to produce dissociative sedation, a cataleptic state of profound analgesia and amnesia. The response to pharmacological agents is highly individual, and the drugs used occasionally have unpredictable pharmacokinetics and pharmacodynamics, which can always result in progression between levels of sedation irrespective of the intention. Therefore, practitioners must be able to ‘rescue’ patients from deeper sedation. These practitioners need to be senior clinicians with advanced airway skills (AAS) training. It can lead to adverse effects such as dangerous hypoxia and unintended loss of consciousness if incorrectly undertaken; studies by the National Confidential Enquiry into Patient Outcome and Death (NCEPOD) have reported avoidable deaths. The Royal College of Emergency Medicine, UK (RCEM) released an updated ‘Safe Sedation of Adults in the Emergency Department’ guidance in 2017 detailing a series of standards for staff competencies, and the required environment and equipment, which are required for each target sedation depth. The emergency department in Jersey undertook audit research in 2018 to assess their current practice. It showed gaps in clinical competency, the need for uniform care, and improved documentation. This spurred the development of a checklist incorporating the above RCEM standards, including contraindication for procedural sedation and difficult airway assessment. This was approved following discussion with the relevant heads of departments and the patient safety directorates. Following this, a second audit research was carried out in 2019 with 17 completed checklists (11 relocation of joints, 6 cardioversions). Data was obtained from looking at the controlled resuscitation drugs book containing documented use of ketamine, alfentanil, and fentanyl. TrakCare, which is the patient electronic record system, was then referenced to obtain further information. The results showed dramatic improvement compared to 2018, and they have been subdivided into six categories; pre-procedure assessment recording of significant medical history and ASA grade (2 fold increase), informed consent (100% documentation), pre-oxygenation (88%), staff (90% were AAS practitioners) and monitoring (92% use of non-invasive blood pressure, pulse oximetry, capnography, and cardiac rhythm monitoring) during procedure, and discharge instructions including the documented return of normal vitals and consciousness (82%). This procedural sedation checklist is a safe intervention that identifies pertinent information about the patient and provides a standardised checklist for the delivery of gold standard of care.

Keywords: advanced airway skills, checklist, procedural sedation, resuscitation

Procedia PDF Downloads 117