Dancing with chemical formulae of antivirals: A panoramic view (Part 2)

In this second part of “Dancing with antivirals as chemical formulae” I will focus on a number of chemical compounds that in the last few years have elicited more than common attraction from a commercial viewpoint: (i) favipiravir (T-705), as it is active against influenza, but also several other RNA viruses; (ii) neuraminidase inhibitors such as zanamivir and oseltamivir; (iii) peramivir and laninamivir octanoate, which might be effective against influenza virus following a single (intravenous or inhalation) administration; (iv) sofosbuvir, the (anticipated) cornerstone for the interferon-free therapy of HCV infections; (v) combinations of DAAs (direct antiviral agents) to achieve, in no time, a sustained virus response (SVR) against HCV infection; (vi) HIV protease inhibitors, the latest and most promising being darunavir; (vii) the integrase inhibitors (INIs) (raltegravir, elvitegravir, dolutegravir), representing a new dimension in the anti-HIV armamentarium; (viii), a new class of helicase primase inhibitors (HPIs) that may exceed acyclovir and the other anti-herpes compounds in both potency and safety; (ix) CMX-001, as the latest of Dr. Antonín Holý’s legacy for its activity against poxviruses and CMV infections, and (x) noroviruses for which the ideal antiviral compounds are still awaited for.     

Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: From ultrapotent to hyperpotent

Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho = 0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC₅₀ values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.     

Mitochondrial dysfunction in cancer chemoresistance

Mitochondrial dysfunction has been associated with cancer development and progression. Recent evidences suggest that pathogenic mutations or depletion of the mitochondrial genome can contribute to development of chemoresistance in malignant tumors. In this review we will describe the current knowledge on the role of mitochondrial dysfunction in the development of chemoresistance in cancer. We will also discuss the significance of this research topic in the context of development of more effective, targeted therapeutic modalities and diagnostic strategies for cancer patients, with a particular focus on the potential use of PARP inhibitors in cancer patients displaying mitochondrial DNA mutations. We will discuss recent studies highlighting the importance of the cross-talk between the tumor microenvironment and mitochondrial functionality in determining selective response to certain chemotherapeutic drugs. Finally, owing to the similarities between cancer and yeast cell metabolism, we will point out the use of yeast as a model system to study cancer-related genes and for anti-cancer drugs screening.     

Angiotensin (1–7) protects against stress-induced gastric lesions in rats

Stress ulcers can develop with severe physiological stress, and have been proposed as being brain-driven events. New findings continue to suggest that stress ulcers can be more effectively managed through central manipulation rather than by simply altering local gastric factors. Angiotensin (1–7) (Ang (1–7)) is present as an endogenous constituent of the brain and stomach. The beneficial effects of Ang (1–7) have been confirmed in the vessels, brain, heart, kidney, liver and lungs, but not in the stomach. Given the accumulating evidence suggesting the anti-stress activities of Ang (1–7), its potential gastroprotective effect in the context of stress requires further investigation. In the present study, rat gastric mucosal lesions were induced by 2 h of cold-restraint stress. We observed that these lesions were significantly attenuated after 1 week of intracerebroventricular treatment with Ang (1–7). This gastroprotective effect was associated with attenuated oxidative stress and suppressed acid secretion. Brain Ang (1–7) administration profoundly modified responses to stress, indicated by altered levels of several stress hormones, including Ang II, glucocorticoid, norepinephrine, serotonin, and dopamine, in blood or stress-related brain regions. These findings indicate that Ang (1–7) exerts anti-stress activities by restoring the gastric microenvironment and modulating the stress pathways. Ang (1–7) may be a promising agent for stress ulcer prophylaxis and therapy, administered through brain-permeable mimics or carriers.     

Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells

Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC.     

Influence of borneol and muscone on geniposide transport through MDCK and MDCK-MDR1 cells as blood–brain barrier in vitro model

The objective of this study was (1) to characterize geniposide transport through MDCK and MDCK-MDR1 cell lines to confirm its transport mechanism and (2) to evaluate the effect of borneol and muscone as enhancers of geniposide transport in the BBB models so as to explore the enhancement mechanism. Transport studies of geniposide were performed in both directions, from apical to basolateral and from basolateral to apical sides. Drug concentrations were analyzed by HPLC. Geniposide showed relatively poor absorption in MDCK and MDCK-MDR1 cells, apparent permeability coefficients ranging from 0.323 × 10⁻⁶ to 0.422 × 10⁻⁶ cm/s. The in vitro experiments showed that geniposide transport in both directions was not concentration dependent and saturable, indicating purely passive diffusion. The efflux ratio of geniposide was less than 2 in the two cell models, which suggested that geniposide was not P-gp substrates. Geniposide transport in both directions significantly increased when co-administrated with increasing concentrations of borneol and muscone. Actin staining results indicated that borneol and muscone increased geniposide transport in the BBB models may attribute to disassembly effect on tight junction integrity.     

Internal mouthpiece designs as a future perspective for enhanced aerosol deposition. Comparative results for aerosol chemotherapy and aerosol antibiotics

Background

In an effort to identify factors producing a finest mist from Jet-Nebulizers we designed 2 mouthpieces with 4 different internal designs and 1–3 compartments.

Materials and methods

Ten different drugs previous used with their “ideal” combination of jet-nebulizer, residual-cup and loading were used. For each drug the mass median aerodynamic diameter size had been established along with their “ideal” combination.

Results

For both mouthpiece, drug was the most important factor due the high F-values (F_large = 251.7, p < 0.001 and F_small = 60.1, p < 0.001) produced. The design affected the droplet size but only for large mouthpiece (F_large = 5.99, p = 0.001, F_small = 1.72, p = 0.178). Cross designs create the smallest droplets (2.271) so differing from the other designs whose mean droplets were greater and equal ranging between 2.39 and 2.447. The number of compartments in the two devices regarding the 10 drugs was found not statistically significant (p-values 0.768 and 0.532 respectively). Interaction effects between drugs and design were statistically significant for both devices (F_large = 8.87, p < 0.001, F_small = 5.33, p < 0.001).

Conclusion

Based on our experiment we conclude that further improvement of the drugs intended for aerosol production is needed. In addition, the mouthpiece design and size play an important role in further enhancing the fine mist production and therefore further experimentation is needed.     

Functional G-protein-coupled receptor 35 is expressed by neurons in the CA1 field of the hippocampus

The G-protein-coupled receptor 35 (GPR35) was de-orphanized after the discovery that kynurenic acid (KYNA), an endogenous tryptophan metabolite, acts as an agonist of this receptor. Abundant evidence supports that GPR35 exists primarily in peripheral tissues. Here, we tested the hypothesis that GPR35 exists in the hippocampus and influences the neuronal activity. Fluorescence immunohistochemical staining using an antibody anti-NeuN (a neuronal marker), an antibody anti-GFAP (a glial marker), and an antibody anti-GPR35 revealed that neurons in the stratum oriens, stratum pyramidale, and stratum radiatum of the CA1 field of the hippocampus express GPR35. To determine the presence of functional GPR35 in the neurocircuitry, we tested the effects of various GPR35 agonists on the frequency of spontaneous action potentials recorded as fast current transients (CTs) from stratum radiatum interneurons (SRIs) under cell-attached configuration in rat hippocampal slices. Bath application of the GPR35 agonists zaprinast (1–10 μM), dicumarol (50–100 μM), pamoic acid (500–1000 μM), and amlexanox (3 μM) produced a concentration- and time-dependent reduction in the frequency of CTs. Superfusion of the hippocampal slices with the GPR35 antagonist ML145 (1 μM) increased the frequency of CTs and reduced the inhibitory effect of zaprinast. Bath application of phosphodiesterase 5 inhibitor sildenafil (1 or 5 μM) was ineffective, whereas a subsequent application of zaprinast was effective in reducing the CT frequency. The present results demonstrate for the first time that functional GPR35s are expressed by CA1 neurons and suggest that these receptors can be molecular targets for controlling neuronal activity in the hippocampus.     

Optical microscopy as a comparative analytical technique for single-particle dissolution studies

Novel, simple and cost effective methods are needed to replace advanced chemical analytical techniques, in small-scale dissolution studies. Optical microscopy of individual particles could provide such a method. The aim of the present work was to investigate and verify the applicability of optical microscopy as an analytical technique for drug dissolution studies. The evaluation was performed by comparing image and chemical analysis data of individual dissolving particles. It was shown that the data obtained by image analysis and UV-spectrophotometry produced practically identical dissolution curves, with average similarity and difference factors above 82 and below 4, respectively. The relative standard deviation for image analysis data, of the studied particle size range, varied between 1.9% and 3.8%. Consequently, it is proposed that image analysis can be used, on its own, as a viable analytical technique in single-particle dissolution studies. The possibility for significant reductions in sample preparation, operational cost, time and substance consumption gives optical detection a clear advantage over chemical analytical methods. Thus, image analysis could be an ideal and universal analytical technique for rapid small-scale dissolution studies.     

Protein–nanoparticle interactions evaluation by immunomethods: Surfactants can disturb quantitative determinations

The adsorption of proteins on nanoparticle surface is one of the first events that occur when nanoparticles enter in the blood stream, which influences nanoparticles lifetime and further biodistribution. Albumin, which is the most abundant protein in serum and which has been deeply characterized, is an interesting model protein to investigate nanoparticle–protein interactions. Therefore, the interaction of nanoparticles with serum albumin has been widely studied. Immunomethods were suggested for the investigation of adsorption isotherms because of their ease to quantify the non-adsorbed bovine serum albumin without the need of applying separation methods that could modify the balance between the adsorbed and non-adsorbed proteins. The present work revealed that this method should be applied with caution. Artifacts in the determination of free protein can be generated by the presence of surfactants such as polysorbate 80, widely used in the pharmaceutical and biomedical field, that are needed to preserve the stability of nanoparticle dispersions. It was shown that the presence of traces of polysorbate 80 in the dispersion leads to an overestimation of the amount of bovine serum albumin remaining free in the dispersion medium when determined by both radial immunodiffusion and rocket immunoelectrophoresis. However, traces of poloxamer 188 did not result in clear perturbed migrations. These methods are not appropriate to perform adsorption isotherms of proteins on nanoparticle dispersions containing traces of remaining free surfactant. They should only be applied on dispersions that are free of surfactant that is not associated with nanoparticles.     

Anti-atherogenic effects of statins: Impact on angiopoietin-2 release from endothelial cells

Beyond lipid lowering, statins are supposed to exert pleiotropic effects positively influencing the progression of atherosclerotic lesions. The development of such lesions is associated with increased release of angiopoietin-2 (Ang-2), an endothelial cell-specific protein growth factor stored in Weibel-Palade bodies (WPBs). The aim of our study was to examine whether statin pretreatment influences the release of Ang-2 from endothelial cells. Stimulation of HUVECs and HMVECs with PMA, thrombin or histamine resulted in significant release of Ang-2, as evidenced by ELISA. Pretreatment with simvastatin and mevastatin suppressed this release to basal level, while pravastatin had no effect. Simvastatin itself increased nitric oxide (NO, EC number 1.14.13.39) synthesis, measured by Griess reaction. Combining the statin pretreatment with the eNOS inhibitor L-NNA as well as bypassing the HMG-CoA reductase (EC number: 1.1.1.34) by adding mevalonic acid or geranyl pyrophosphate restored the exocytotic effect of PMA. Immunofluorescence microscopy showed that depletion of WPBs upon PMA stimulation ceased after pretreatment with simvastatin. This study demonstrates a potent suppressive effect of statins on the release of Ang-2 from endothelial cells. Regarding its harmful effects in the development of atherosclerotic lesions, our data provide further insight into the mechanisms of the anti-atherogenic potential of statins.     

The effect of polymer properties on direct compression and drug release from water-insoluble controlled release matrix tablets

The objective of this study was to identify and evaluate key polymer properties affecting direct compression and drug release from water-insoluble matrices. Commonly used polymers, such as Kollidon^® SR, Eudragit^® RS and ethyl cellulose, were characterized, formulated into tablets and compared with regard to their properties in dry and wet state. A similar site percolation threshold of 65% v/v was found for all polymers in dry state. Key parameters influencing polymer compactibility were the surface properties and the glass transition temperature (T_g), affecting polymer elasticity and particle size-dependent binding. The important properties observed in dry state also governed matrix characteristics and therefore drug release in wet state. A low T_g (Kollidon^® SR < Eudragit^® RS) decreased the percolation threshold, particle size effect and tortuosity, but increased permeability and sensitivity to heat/humidity treatment. Hence, lower permeability and higher stability are benefits of a high-T_g polymer (ethyl cellulose). However, release retardation was observed in the same order as matrix integrity (Eudragit^® RS < ethyl cellulose < Kollidon^® SR), as the high permeability was counteracted by PVP in case of Kollidon^® SR. Therefore, the T_g and composition of a polymer need to be considered in polymer design and formulation of controlled-release matrix systems.     

Chronic oleoylethanolamide treatment improves spatial cognitive deficits through enhancing hippocampal neurogenesis after transient focal cerebral ischemia

Oleoylethanolamide (OEA) has been shown to have neuroprotective effects after acute cerebral ischemic injury. The aim of this study was to investigate the effects of chronic OEA treatment on ischemia-induced spatial cognitive impairments, electrophysiology behavior and hippocampal neurogenesis. Daily treatments of 30 mg/kg OEA significantly ameliorated spatial cognitive deficits and attenuated the inhibition of long-term potentiation (LTP) in the middle cerebral artery occlusion (MCAO) rat model. Moreover, OEA administration improved cognitive function in a manner associated with enhanced neurogenesis in the hippocampus. Further study demonstrated that treatment with OEA markedly increased the expressions of brain-derived neurotrophic factor (BDNF) and peroxisome proliferator-activated receptors α (PPARα). Our data suggest that chronic OEA treatment can exert functional recovery of cognitive impairments and neuroprotective effects against cerebral ischemic insult in rats via triggering of neurogenesis in the hippocampus, which supports the therapeutic use of OEA for cerebral ischemia.     

Acyclovir chemical kinetics with the discovery and identification of newly reported degradants and degradation pathways involving formaldehyde as a degradant and reactant intermediate

The purpose of this research was to determine acyclovir (ACV) acidic degradation kinetics which is relevant to gastric retentive device product design. A stability-indicating method revealed two unknown degradation products which have been identified by mass spectrometry as ACV and guanine formaldehyde adducts. In addition to the formation of these adducts, a proposed degradation scheme identifies the formation of methyl acetal ethylene glycol, formaldehyde, ethylene glycol, and guanine as additional ACV degradation products. pH-rate profiles were explained by using a rate law which assumed acid-catalyzed hydrolysis of protonated and unprotonated ACV. The predicted and observed rate constants were in good agreement. Data-driven excipient selection recommendations were based on the chemical kinetic study results, degradation scheme, and pH-rate profiles. The average activation energy for the degradation reaction was determined to be 31.3 ± 1.6 kcal/mol. The predicted ACV t_90% at 37 °C and pH 1.2 was calculated to be 7.2 days. As a first approximation, this suggests that ACV gastric retentive devices designed to deliver drug for 7 days should have acceptable drug product stability in the stomach.     

Protective role of amantadine in mitochondrial dysfunction and oxidative stress mediated by hepatitis C virus protein expression

Amantadine is an antiviral and antiparkinsonian drug that has been evaluated in combination therapies against hepatitis C virus (HCV) infection. Controversial results have been reported concerning its efficacy, and its mechanism of action remains unclear. Data obtained in vitro suggested a role of amantadine in inhibiting HCV p7-mediated cation conductance. In keeping with the fact that mitochondria are responsible to ionic fluxes and that HCV infection impairs mitochondrial function, we investigated a potential role of amantadine in modulating mitochondrial function. Using a well-characterized inducible cell line expressing the full-length HCV polyprotein, we found that amantadine not only prevented but also rescued HCV protein-mediated mitochondrial dysfunction. Specifically, amantadine corrected (i) overload of mitochondrial Ca²⁺; (ii) inhibition of respiratory chain activity and oxidative phosphorylation; (iii) reduction of membrane potential; and (iv) overproduction of reactive oxygen species. The effects of amantadine were observed within 15 min following drug administration and confirmed in Huh-7.5 cells transfected with an infectious HCV genome. These effects were also observed in cells expressing subgenomic HCV constructs, indicating that they are not mediated or only in part mediated by p7. Single organelle analyzes carried out on isolated mouse liver mitochondria demonstrated that amantadine induces hyperpolarization of the membrane potential. Moreover, amantadine treatment increased the calcium threshold required to trigger mitochondrial permeability transition opening. In conclusion, these results support a role of amantadine in preserving cellular bioenergetics and redox homeostasis in HCV-infected cells and unveil an effect of the drug which might be exploited for a broader therapeutic utilization.     

Blood–brain barrier models and their relevance for a successful development of CNS drug delivery systems: A review

During the research and development of new drugs directed at the central nervous system, there is a considerable attrition rate caused by their hampered access to the brain by the blood–brain barrier. Throughout the years, several in vitro models have been developed in an attempt to mimic critical functionalities of the blood–brain barrier and reliably predict the permeability of drug candidates. However, the current challenge lies in developing a model that retains fundamental blood–brain barrier characteristics and simultaneously remains compatible with the high throughput demands of pharmaceutical industries. This review firstly describes the roles of all elements of the neurovascular unit and their influence on drug brain penetration. In vitro models, including non-cell based and cell-based models, and in vivo models are herein presented, with a particular emphasis on their methodological aspects. Lastly, their contribution to the improvement of brain drug delivery strategies and drug transport across the blood–brain barrier is also discussed.     

The total margin of exposure of ethanol and acetaldehyde for heavy drinkers consuming cider or vodka

Heavy drinkers in Scotland may consume 1600 g ethanol per week. Due to its low price, cider may be preferred over other beverages. Anecdotal evidence has linked cider to specific health hazards beyond other alcoholic beverages. To examine this hypothesis, nine apple and pear cider samples were chemically analysed for constituents and contaminants. None of the products exceeded regulatory or toxicological thresholds, but the regular occurrence of acetaldehyde in cider was detected. To provide a quantitative risk assessment, two collectives of exclusive drinkers of cider and vodka were compared and the intake of acetaldehyde was estimated using probabilistic Monte–Carlo type analysis. The cider consumers were found to ingest more than 200-times the amount of acetaldehyde consumed by vodka consumers. The margins of exposure (MOE) of acetaldehyde were 224 for the cider and over 220,000 for vodka consumers. However, if the effects of ethanol were considered in a cumulative assessment of the combined MOE, the effect of acetaldehyde was minor and the combined MOE for both groups was 0.3. We suggest that alcohol policy priority should be given on reducing ethanol intake by measures such as minimum pricing, rather than to focus on acetaldehyde.     

In-vitro and in-vivo cytotoxicity and efficacy evaluation of novel glycyl-glycine and alanyl-alanine conjugates of chitosan and trimethyl chitosan nano-particles as carriers for oral insulin delivery

Purpose

The aim of this research work was to explore the possibility of providing multifunctional oral insulin delivery system by conjugating several types of dipeptides on chitosan and trimethyl chitosan to be used as drug carriers.