Pharmaceuticals — Open Access Pharmaceutical Sciences Journal

Tumor suppressor genes can be silenced genetically as well as epigenetically. One approach to reversing epigenetic suppression of tumor suppressor genes is to inhibit DNA methyl transferase. 5-aza-2′,2′-diflurorodeoxycytidine (NUC013) is a novel DNA methyl transferase and ribonucleotide reductase inhibitor that is a more potent inhibitor of growth than decitabine in the NCI 60 cancer cell line panel. NUC013 is more active than decitabine against p53-null/mutant cancer cell lines (p = 0.027) but is even more so against p53 wild-type (WT) cell lines (p = 0.0025). The maximum tolerated dose in mice of NUC013 is greater than 120 mg/kg administered intravenously for three consecutive days a week for three weeks. With this regimen and a dose of 20 mg/kg in a human leukemia HL-60 (p53-null) NCr-nu/nu mouse xenograft model (n = 10/group), NUC013 demonstrated a survival benefit (saline median survival (MS) = 26.5 days, NUC013 MS = 32 days and hazard ratio (HR) = 0.26 (p = 0.032)). In a colon cancer LoVo (TP53 WT) xenograft, mice treated with decitabine at 5 mg/kg had worse survival than saline controls (decitabine MS = 31 days, saline MS > 60 days and HR = 26.89 (p < 0.0001)). At a dose of 20 mg/kg NUC013, mean tumor volume in the LoVo xenografts was lower than controls by 50.9% and at 40 mg/kg by 53.7% (both p < 0.0001). Full article

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders and is characterized by the loss of dopaminergic neurons in the substantia nigra (SN). Although the causes of PD are not understood, evidence suggests that its pathogenesis is associated with oxidative stress and inflammation. Recent studies have suggested a protective role of the cannabinoid signalling system in PD. β-caryophyllene (BCP) is a natural bicyclic sesquiterpene that is an agonist of the cannabinoid type 2 receptor (CB2R). Previous studies have suggested that BCP exerts prophylactic and/or curative effects against inflammatory bowel disease through its antioxidative and/or anti-inflammatory action. The present study describes the neuroprotective effects of BCP in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced murine model of PD, and we report the results of our investigation of its neuroprotective mechanism in neurons and glial cells. In the murine model, BCP pretreatment ameliorated motor dysfunction, protected against dopaminergic neuronal losses in the SN and striatum, and alleviated MPTP-induced glia activation. Additionally, BCP inhibited the levels of inflammatory cytokines in the nigrostriatal system. The observed neuroprotection and inhibited glia activation were reversed upon treatment with the CB2R selective antagonist AM630, confirming the involvement of the CB2R. These results indicate that BCP acts via multiple neuroprotective mechanisms in our murine model and suggest that BCP may be viewed as a potential treatment and/or preventative agent for PD. Full article

2017 marks the 20th anniversary of the molecular cloning by David Julius and colleagues (1997) of the long sought-after capsaicin receptor, now known as TRPV1 (Transient Receptor Potential Vanilloid 1) [1]. This seminal discovery has opened up a “hot” new field of basic research and launched drug discovery efforts into the large family (by the latest count 28 mammalian members, 27 in humans) of TRP ion channels [2]. Indeed, it took less than a decade for the first potent, small molecule TRPV1 antagonists to enter phase 1 clinical trials [3]. Yet, despite the large amount of resources that has been invested in TRPV1 research, there are currently no TRPV1-targeted drugs in phase 3 clinical trials. In this special issue of Pharmaceuticals, we aim to capture the progress in the TRP channel field over the past twenty years, with 15 articles covering a variety of TRP channels and potential relevant disease states and applications. Full article

The synthesis and biological evaluation of a series of novel heterocyclic indole derivatives is described. The consolidation of the combretastatin and bisindolylmaleimide templates towards the inclusion of a novel heterocyclic ring proffered a versatile pharmacophore with which to pursue chemical diversification. Given literature precedent, maleimide was initially investigated in this role and the bioactivity assessed by measurement of NCI-60 cell panel growth. Subsequently, a range of 5-aminopyrazoles was designed and developed to explore the specific effect of heterocycle hydrogen bonding on cell growth. The unique electronic nature of the 5-aminopyrazole moiety allowed for regiospecific monosubstitution on different sites of the ring, such as thiourea substitution at the N(1) position for derivative 45 or trifluoroacetylation on the 5-amino position for 43. Further derivatisation led to the ultimate development of bicyclic pyrazolotriazinedione 41 and pyrimidine 42 systems. The antiproliferative activities of these 3,4-diaryl-5-aminopyrazoles were assessed using the NCI-60 cell screen, disclosing the discovery of distinct selectivity profiles towards a number of cell lines, such as SNB-75 CNS cancer, UO-31 and CAKI-1 renal cancer cells. A series of DNA topological assays discounted the interaction with topoisomerase II as a putative mechanism of action. Full article

The complex pathophysiology of spinal cord injury (SCI) may explain the current lack of an effective therapeutic approach for the regeneration of damaged neuronal cells and the recovery of motor functions. A primary mechanical injury in the spinal cord triggers a cascade of secondary events, which are involved in SCI instauration and progression. The aim of the present review is to provide an overview of the therapeutic neuro-protective and neuro-regenerative approaches, which involve the use of nanofibers as local drug delivery systems. Drugs released by nanofibers aim at preventing the cascade of secondary damage (neuro-protection), whereas nanofibrous structures are intended to re-establish neuronal connectivity through axonal sprouting (neuro-regeneration) promotion, in order to achieve a rapid functional recovery of spinal cord. Full article

Current therapeutic unfractionated heparin available in Europe and US is of porcine mucosal origin. There is now interest, specifically in the US, to use bovine mucosa as an additional source for the production of heparin. The anticoagulant action of heparin can be neutralized by protamine sulfate, and in this study the ability of protamine to bind and neutralize the anticoagulant activities of heparin from porcine mucosa, bovine mucosa and bovine lung were assessed. Protamine sulfate was able to bind and precipitate similar amounts of heparins from different sources on a mass basis. However, differential amounts of anticoagulant activities were neutralized by protamine sulfate, with neutralization of porcine mucosa more effective than for bovine lung and bovine mucosa. For all heparins, potentiation of thrombin inhibition by antithrombin and heparin cofactor II was preferentially neutralized over antithrombin-mediated inhibition of factor Xa or plasma clotting time. Whole blood thromboelastography showed that neutralization by protamine sulfate was more effective than the antithrombin dependent thrombin inhibition assays indicated. While there was no absolute correlation between average or peak molecular weight of heparin samples and neutralization of anticoagulant activity, correlation was observed between proportions of material with high affinity to antithrombin, specific activities and neutralization of activity. Full article

Pseudomonas aeruginosa is involved in a variety of difficult-to-treat infections frequently due to biofilm formation. To identify useful antibiofilm strategies, this article evaluated efficacy of two newly engineered cationic antimicrobial peptides (17BIPHE2 and DASamP2), traditional antibiotics, and their combinations against biofilms at different stages. 17BIPHE2 is designed based on the 3D structure of human cathelicidin LL-37 and DASamP2 is derived from database screening. While both peptides show effects on bacterial adhesion, biofilm formation, and preformed biofilms, select antibiotics only inhibit biofilm formation, probably due to direct bacterial killing. In addition, the time dependence of biofilm formation and treatment in a static in vitro biofilm model was also studied. The initial bacterial inoculum determines the peptide concentration needed to inhibit biofilm growth. When the bacterial growth time is less than 8 h, the biomass in the wells can be dispersed by either antibiotics alone or peptides alone. However, nearly complete biofilm disruption can be achieved when both the peptide and antibiotics are applied. Our results emphasize the importance of antibiofilm peptides, early treatment using monotherapy, and the combination therapy for already formed biofilms of P. aeruginosa. Full article

The molecular mechanisms underlying tissue regeneration and wound healing are still poorly understood despite their importance. In this paper we develop a bioinformatics approach, combining biology and network theory to drive experiments for better understanding the genetic underpinnings of wound healing mechanisms and for selecting potential drug targets. We start by selecting literature-relevant genes in murine wound healing, and inferring from them a Protein-Protein Interaction (PPI) network. Then, we analyze the network to rank wound healing-related genes according to their topological properties. Lastly, we perform a procedure for in-silico simulation of a treatment action in a biological pathway. The findings obtained by applying the developed pipeline, including gene expression analysis, confirms how a network-based bioinformatics method is able to prioritize candidate genes for in vitro analysis, thus speeding up the understanding of molecular mechanisms and supporting the discovery of potential drug targets. Full article

Folate receptor (FR)-targeted radionuclide therapy using folate radioconjugates is of interest due to the expression of the FR in a variety of tumor types. The high renal accumulation of radiofolates presents, however, a risk of radionephropathy. A potential option to address this challenge would be to use radioprotectants, such as amifostine. Methods for early detection of kidney damage that—in this case—cannot be predicted based on dose estimations, would facilitate the development of novel therapies. The aim of this study was, therefore, to assess potentially changing levels of plasma and urine biomarkers and to determine DNA damage at an early stage after radiofolate application. The identification of an early indicator for renal damage in mice would be useful since histological changes become apparent only several months after treatment. Mice were injected with different quantities of ¹⁷⁷Lu-folate (10 MBq, 20 MBq and 30 MBq), resulting in mean absorbed kidney doses of ~23 Gy, ~46 Gy and ~69 Gy, respectively, followed by euthanasia two weeks (>85% of the mean renal radiation dose absorbed) or three months later. Whereas all investigated biomarkers remained unchanged, the number of γ-H2AX-positive nuclei in the renal cortex showed an evident dose-dependent increase as compared to control values two weeks after treatment. Comparison with the extent of kidney injury determined by histological changes five to eight months after administration of the same ¹⁷⁷Lu-folate activities suggested that the quantitative assessment of double-strand breaks can be used as a biological indicator for long-term radiation effects in the kidneys. This method may, thus, enable faster assessment of radiopharmaceuticals and protective measures by preventing logistically challenging long-term investigations to detect kidney damage. Full article

In the context of radiopharmacy and molecular imaging, the concept of theranostics entails a therapy-accompanying diagnosis with the aim of a patient-specific treatment. Using the adequate diagnostic radiopharmaceutical, the disease and the state of the disease are verified for an individual patient. The other way around, it verifies that the radiopharmaceutical in hand represents a target-specific and selective molecule: the “best one” for that individual patient. Transforming diagnostic imaging into quantitative dosimetric information, the optimum radioactivity (expressed in maximum radiation dose to the target tissue and tolerable dose to healthy organs) of the adequate radiotherapeutical is applied to that individual patient. This theranostic approach in nuclear medicine is traced back to the first use of the radionuclide pair ⁸⁶Y/⁹⁰Y, which allowed a combination of PET and internal radiotherapy. Whereas the β-emitting therapeutic radionuclide ⁹⁰Y (t½ = 2.7 d) had been available for a long time via the ⁹⁰Sr/⁹⁰Y generator system, the β⁺ emitter ⁸⁶Y (t½ = 14.7 h) had to be developed for medical application. A brief outline of the various aspects of radiochemical and nuclear development work (nuclear data, cyclotron irradiation, chemical processing, quality control, etc.) is given. In parallel, the paper discusses the methodology introduced to quantify molecular imaging of ⁸⁶Y-labelled compounds in terms of multiple and long-term PET recordings. It highlights the ultimate goal of radiotheranostics, namely to extract the radiation dose of the analogue ⁹⁰Y-labelled compound in terms of mGy or mSv per MBq ⁹⁰Y injected. Finally, the current and possible future development of theranostic approaches based on different PET and therapy nuclides is discussed. Full article

Cartilage defects represent a serious problem due to the poor regenerative properties of this tissue. Regarding the nose, nasal valve collapse is associated with nasal blockage and persistent airway obstruction associated with a significant drop in the quality of life for patients. In addition to surgical techniques, several cell-based tissue-engineering strategies are studied to improve cartilage support in the nasal wall, that is, to ameliorate wall insufficiency. Nevertheless, there are no congruent data available on the benefit for patients during the follow-up time. In this manuscript, we propose an innovative approach in the treatment of cartilage defects in the nose (nasal valve collapse) based on autologous micro-grafts obtained by mechanical disaggregation of a small portion of cartilage tissue (Rigenera^® protocol). In particular, we first analyzed in vitro murine and human cartilage micro-grafts; secondly, we analyzed the clinical results of a patient with pinched nose deformity treated with autologous micro-grafts of chondrocytes obtained by Rigenera^® protocol. The use of autologous micro-graft produced promising results in surgery treatment of cartilage injuries and could be safely and easily administrated to patients with cartilage tissue defects. Full article

High sulfation, low cost, and the status of heparin as an already FDA- and EMA- approved product, mean that its inclusion in tissue engineering (TE) strategies is becoming increasingly popular. However, the use of heparin may represent a naïve approach. This is because tissue formation is a highly orchestrated process, involving the temporal expression of numerous growth factors and complex signaling networks. While heparin may enhance the retention and activity of certain growth factors under particular conditions, its binding ‘promiscuity’ means that it may also inhibit other factors that, for example, play an important role in tissue maintenance and repair. Within this review we focus on articular cartilage, highlighting the complexities and highly regulated processes that are involved in its formation, and the challenges that exist in trying to effectively engineer this tissue. Here we discuss the opportunities that glycosaminoglycans (GAGs) may provide in advancing this important area of regenerative medicine, placing emphasis on the need to move away from the common use of heparin, and instead focus research towards the utility of specific GAG preparations that are able to modulate the activity of growth factors in a more controlled and defined manner, with less off-target effects. Full article

In an ongoing research program for the development of new anti-tuberculosis drugs, we synthesized three series (A, B, and C) of 7-chloro-4-aminoquinolines, which were evaluated in vitro against Mycobacterium tuberculosis (MTB). Now, we report the anti-MTB and cytotoxicity evaluations of a new series, D (D01–D21). Considering the active compounds of series A (A01–A13), B (B01–B13), C (C01–C07), and D (D01–D09), we compose a data set of 42 compounds and carried out hologram quantitative structure–activity relationship (HQSAR) analysis. The amino–imino tautomerism of the 4-aminoquinoline moiety was considered using both amino (I) and imino (II) forms as independent datasets. The best HQSAR model from each dataset was internally validated and both models showed significant statistical indexes. Tautomer I model: leave-one-out (LOO) cross-validated correlation coefficient (q²) = 0.80, squared correlation coefficient (r²) = 0.97, standard error (SE) = 0.12, cross-validated standard error (SE_cv) = 0.32. Tautomer II model: q² = 0.77, r² = 0.98, SE = 0.10, SE_cv = 0.35. Both models were externally validated by predicting the activity values of the corresponding test set, and the tautomer II model, which showed the best external prediction performance, was used to predict the biological activity responses of the compounds that were not evaluated in the anti-MTB trials due to poor solubility, pointing out D21 for further solubility studies to attempt to determine its actual biological activity. Full article

Tuberculosis (TB), a disease caused mainly by the Mycobacterium tuberculosis (Mtb), is according to the World Health Organization (WHO) the infectious disease responsible for the highest number of deaths worldwide. The increased number of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains, and the ineffectiveness of the current treatment against latent tuberculosis are challenges to be overcome in the coming years. The scenario of drug discovery becomes alarming when it is considered that the number of new drugs does not increase proportionally to the emergence of drug resistance. In this review, we will demonstrate the current advances in antitubercular drug discovery, focusing on the research of compounds with potent antituberculosis activity against MDR-TB strains. Herein, active compounds against MDR-TB with minimum inhibitory concentrations (MICs) less than 11 µM and low toxicity published in the last 4 years in the databases PubMed, Web of Science and Scopus will be presented and discussed. Full article

Searching of new enantiomerically pure chiral derivatives of xanthones (CDXs) with potential pharmacological properties, particularly those with anti-inflammatory activity, has remained an area of interest of our group. Herein, we describe in silico studies and in vitro inhibitory assays of cyclooxygenases (COX-1 and COX-2) for different enantiomeric pairs of CDXs. The evaluation of the inhibitory activities was performed by using the COX Inhibitor Screening Assay Kit. Docking simulations between the small molecules (CDXs; known ligands and decoys) and the enzyme targets were undertaken with AutoDock Vina embedded in PyRx—Virtual Screening Tool software. All the CDXs evaluated exhibited COX-1 and COX-2 inhibition potential as predicted. Considering that the (S)-(−)-enantiomer of the nonsteroidal anti-inflammatory drug ketoprofen preferentially binds to albumin, resulting in lower free plasma concentration than (R)-(+)-enantiomer, protein binding affinity for CDXs was also evaluated by spectrofluorimetry as well as in in silico. For some CDXs enantioselectivity was observed. Full article

Dysregulated expression or activation of matrix metalloproteinases (MMPs) is observed in many kinds of live-threatening diseases. Therefore, MMP imaging for example with radiolabelled MMP inhibitors (MMPIs) potentially represents a valuable tool for clinical diagnostics using non-invasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. This work includes the organic chemical syntheses and in vitro evaluation of five iodinated barbiturate based MMPIs and the selection of derivative 9 for radiosyntheses of isotopologues [¹²³I]9 potentially useful for MMP SPECT imaging and [¹²⁴I]9 for MMP PET imaging. Full article

Effective therapies for chronic or non-healing wounds are still lacking. These tissue insults often result in severe clinical complications (i.e., infections and/or amputation) and sometimes lead to patient death. Accordingly, several research groups have focused their efforts in finding innovative and powerful therapeutic strategies to overcome these issues. On the basis of these considerations, the comprehension of the molecular cascades behind these pathological conditions could allow the identification of molecules against chronic wounds. In this context, the regulation of the Protein Kinase C (PKC) cascade has gained relevance in the prevention and/or reparation of tissue damages. This class of phosphorylating enzymes has already been considered for different physiological and pathological pathways and modulation of such enzymes may be useful in reparative processes. Herein, the recent developments in this field will be disclosed, highlighting the pivotal role of PKC α and δ in regenerative medicine. Moreover, an overview of well-established PKC ligands, acting via the modulation of these isoenzymes, will be deeply investigated. This study is aimed at re-evaluating widely known PKC modulators, currently utilized for treating other diseases, as fruitful molecules in wound-healing. Full article

Esters based on mono- and bicyclic terpenoids with glycine have been synthesized via Steglich esterification and characterized by ¹H-NMR, IR, and mass spectral studies. Their analgesic and anti-inflammatory activities were investigated after transdermal delivery on models of formalin, capsaicin, and AITC-induced pain, respectively. Glycine esters of menthol and borneol exhibited higher antinociceptive action, whereas eugenol derivative significantly suppressed the development of the inflammatory process. The mechanism of competitive binding between terpenoid esters and TRPA1/TRPV1 agonists was proposed explaining significant analgesic effect of synthesized derivatives. For an explanation of high anti-inflammatory activity, competitive inhibition between terpenoid esters and AITC for binding sites of the TRPA1 ion channel has been suggested. Full article