Falcipain‐2 inhibitors

Malaria, particularly that one caused by Plasmodium falciparum, remains a serious health problem in Africa, South America, and many parts of Asia where it afflicts about 500 million people and is responsible for the death of more than one million children each year. The main reasons for the persistence of malaria are the emergence of resistance to common antimalarial drugs, inadequate control of mosquito vectors, and the lack of effective vaccines. Therefore, the identification and characterization of new targets for antimalarial chemotherapy are of urgent priority. This review is focused on inhibitors of falcipain‐2, a cysteine protease from P. falciparum, which represents one of the most promising targets for antimalarial drug design. Falcipain‐2 is a key enzyme in the life cycle of P. falciparum since it degrades hemoglobin, at the early trophozoite stage, and cleaves ankyrin and protein 4.1, the cytoskeletal elements vital to the stability of red cell membrane, at the schizont stage. The main classes of falcipain‐2 inhibitors are peptides or peptidomimetics bearing the most popular pharmacophores of cysteine protease inhibitors, such as vinyl sulfones, halomethyl ketones, and aldehydes. Furthermore, many other chemotypes have been identified as inhibitors of falcipain‐2, such as isoquinolines, thiosemicarbazones, and chalcones. These inhibitors represent all classes, which, to the best of our knowledge, have been disclosed in journal articles to date. © 2009 Wiley Periodicals, Inc. Med Res Rev, 30, No. 1, 136–167, 2010     

Biological activity of extracellular matrix‐associated BMP‐2

The critical requirement for matrix‐associated bone morphogenetic proteins (BMPs) during induction of bone formation in vivo has long been recognized. However, the role of extracellular matrix (ECM) physisorbed BMPs in inducing the differentiation of resident mesenchymal stem cells into osteoblasts has been ill‐defined. We therefore used BMP‐responsive C2C12s to study the biological activity of collagen type I physisorbed BMP‐2. Fibrillar collagen type I scaffolds were loaded with 75 ng BMP‐2/µg collagen. Under cell culture conditions, 40% of loaded ¹²⁵I‐labelled BMP‐2 was released within 24 h, whereas the remaining BMP‐2 was stably physisorbed for > 7 days. Using these systems suggested that physisorbed BMP‐2 is more active than diffusible BMP‐2. Thus, the current clinical practice of immobilizing BMPs on collagen type I scaffolds not only prolongs local delivery of the morphogen but could also enhance biological activity at the cellular level. Copyright © 2009 John Wiley & Sons, Ltd.     

Dapsone‐induced agranulocytosis—possible involvement of low‐activity N‐acetyltransferase 2

Dapsone‐induced agranulocytosis is a rare but potentially fatal adverse drug reaction (ADR). A 45‐year‐old male Caucasian patient developed agranulocytosis caused by dapsone (diamino‐diphenyl sulfone), which he was prescribed for leukocytoclastic vasculitis. Patient's treatment consisted of termination of dapsone, antibiotic therapy, and granulocyte colony‐stimulating factor leading to prompt improvement of symptoms and normalization of laboratory blood values. Diagnostic evaluation revealed methemoglobinemia and excluded glucose‐6‐phosphate dehydrogenase deficiency. Pharmacogenetics testing showed that he was a carrier of NAT2 *5/*6 genotype, predisposing to low activity of the N‐acetyltransferase 2 enzyme. This was the first and only ADR to dapsone reported in Croatia. In total, there have been 73 ADR to dapsone recorded worldwide, including only four cases of agranulocytosis.     

Endothelium‐dependent and endothelium‐independent effects of 1‐nitro‐2‐propylbenzene on rat aorta

A group of nitro compounds contains a benzene ring in a short aliphatic chain with the NO₂ group, property that supposedly favors its vasodilator profile. In this study, we evaluated in isolated rat aorta the effects of 1‐nitro‐2‐propylbenzene (NPB), a nitro compound containing the NO₂ in the aromatic ring. In aorta precontracted with KCl, NPB (1‐3000 μm ) induced full endothelium‐independent relaxation. In endothelium‐intact preparations, phenylephrine‐induced contractions were fully relaxed by NPB, effect unaltered by N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME) or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ). In the concentration range of 30–300 μm , NPB slightly but significantly potentiated the phenylephrine‐induced contraction. Such potentiation was unaltered by the thromboxane‐prostanoid receptor antagonist seratrodast, but was abolished by endothelium removal or by preincubation of endothelium‐intact preparations with L‐NAME, ODQ or by ruthenium red and HC‐030031, blockers of subtype 1 of ankyrin transient receptor potential (TRPA₁) channels. Verapamil exacerbated the potentiating effect of NPB. The potentiating effect was undetectable in preparations precontracted by 9,11‐dideoxy‐11α,9α‐epoxymethanoprostaglandin F2α (U‐46619). Relaxation was reduced by ruthenium red while it was enhanced by HC‐030031. In conclusion, NPB has vasodilator properties but with a mechanism of action distinct from its analogues. Contrary to other nitro compounds, its relaxing effects did not involve recruitment of the guanylyl cyclase pathway. NPB has also endothelium‐dependent potentiating properties on phenylephrine‐induced contractions, a phenomenon that putatively required a role of endothelial TRPA₁ channels. The present findings reinforce the notion that the functional group NO₂ in the aliphatic chain of these nitro compounds determines favorably their vasodilator properties.     

Effects of transgene‐mediated endomorphin‐2 in inflammatory pain

We examined the analgesic properties of endomorphin‐2 expressed in DRG neurons transduced with a non‐replicating herpes simplex virus (HSV)‐based vector containing a synthetic endomorphin‐2 gene construct. HSV‐mediated endomorphin‐2 expression reduced nocisponsive behaviors in response to mechanical and thermal stimuli after injection of complete Freund's adjuvant (CFA) into the paw, and reduced peripheral inflammation measured by paw swelling after injection of CFA. The analgesic effect of the vector was blocked by either intraperitoneal or intrathecal administration of naloxone methiodide, blocking peripheral and central μ opioid receptors, respectively. Endomorphin‐2 vector injection also reduced spontaneous pain‐related behaviors in the delayed phase of the formalin test and in both CFA and formalin models suppressed spinal c‐fos expression. The magnitude of the vector‐mediated analgesic effect on the delayed phase of the formalin test was similar in naïve animals and in animals with opiate tolerance induced by twice daily treatment with morphine, suggesting that there was no cross‐tolerance between vector‐mediated endomorphin‐2 and morphine. These results suggest that transgene‐mediated expression of endomorphin‐2 in transduced DRG neurons in vivo acts both peripherally and centrally through mu opioid receptors to reduce pain perception.     

(E)‐2‐benzylidene‐4‐phenyl‐1,3‐diselenole has antioxidant and hepatoprotective properties against oxidative damage induced by 2‐nitropropane in rats

The in vitro effect of (E)‐2‐benzylidene‐4‐phenyl‐1,3‐diselenole (BPD) was evaluated through iron/EDTA‐induced thiobarbituric acid reactive species (TBARS) and reactive species (RS) determinations as well as of the scavenging 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH) radical quantification. BPD at the concentrations of 10 and 50 μΜ decreased RS and TBARS levels, respectively. The antioxidant activity was not related to the scavenging DPPH radical mechanism. A second objective of this study was to investigate the hepatoprotective action of BPD, administered by oral route, against oxidative damage induced by 2‐nitropropane (2‐NP) (100 mg/kg of body weight) in liver of rats. At the dose of 50 mg/kg, BPD protected against the increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities induced by 2‐NP. BPD (10 and 50 mg/kg) protected against the increase in TBARS levels and alkaline phosphatase (ALP) activity. Sections of liver from 2‐NP‐exposed rats presented intense infiltration of inflammatory cells and loss of cellular architecture. BPD (10 and 50 mg/kg) attenuated 2‐NP‐induced hepatic histological alterations. The inhibition of δ‐aminolevulinic dehydratase (δ‐ALA‐D), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S‐transferase (GST) activities and the decreased GSH levels caused by 2‐NP were protected by BPD (50 mg/kg). Catalase activity and ascorbic acid levels were not altered by 2‐NP. These results demonstrated the antioxidant and hepatoprotective effects of BPD in liver of rats.     

MR molecular imaging of HER‐2 in a murine tumor xenograft by SPIO labeling of anti‐HER‐2 affibody

In vivo molecular imaging is a rapidly growing research area both for basic and clinical science. Non‐invasive imaging of in vivo conditions at the molecular level increases understanding of the biological characteristics of normal and diseased tissues without the need for invasive surgical procedures. Among the various imaging modalities, magnetic resonance imaging (MRI) has garnered interest as a molecular imaging modality due to its high spatial resolution. Here, we have demonstrated that the combined use of HER‐2 targeting affibody, a small 7 kDa molecule that behaves similarly to antibodies, and superparamagnetic iron oxide (SPIO) can non‐invasively image HER‐2 expressing cells or tissues both in vitro and in vivo by MRI. This preliminary study demonstrates that affibody‐SPIO is a feasible, target‐specific contrast agent for in vivo MR molecular imaging. Copyright © 2010 John Wiley & Sons, Ltd.     

Preliminary evaluation of novel 68Ga‐DOTAVAP‐PEG‐P2 peptide targeting vascular adhesion protein‐1

Introduction: Expression of vascular adhesion protein‐1 (VAP‐1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP‐1 is a potential target for anti‐inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP‐1‐targeting peptide as a potential PET imaging agent. Methods: Cyclic 17‐amino‐acid peptide selected from phage display libraries was 1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetraacetic acid (DOTA) conjugated via 8‐amino‐3,6‐diooxaoctanoyl linker (polyethylene glycol, PEG derivative) and labelled with ⁶⁸Ga (⁶⁸Ga‐DOTAVAP‐PEG‐P2). In vitro stability of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was determined in saline, rat plasma and human plasma by radio‐HLPC. Lipophilicity was measured by calculating octanol‐water partition coefficient (logP). Whole‐body distribution kinetics and stability after intravenous injection in healthy rats was studied in vivo by PET imaging, ex vivo by measuring radioactivity of excised tissues, and by radio‐HPLC. Results: In vitro the ⁶⁸Ga‐DOTAVAP‐PEG‐P2 remained stable >4 h in saline and rat plasma, but degraded slowly in human plasma after 2 h of incubation. The logP value of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was ?1·3. In rats, ⁶⁸Ga‐radioactivity cleared rapidly from blood circulation and excreted quickly in urine. At 120 min after injection the fraction of intact ⁶⁸Ga‐DOTAVAP‐PEG‐P2 were 77 ± 6·0% and 99 ± 1·0% in rat plasma and urine, respectively. Conclusions: These basic and essential in vitro and in vivo studies of the new VAP‐1 targeting peptide revealed promising properties for an imaging agent. Further investigations to clarify in vivo VAP‐1 targeting are warranted.     

Mesenchymal stem cell expression of SDF‐1β synergizes with BMP‐2 to augment cell‐mediated healing of critical‐sized mouse calvarial defects

Bone has the potential for spontaneous healing. This process, however, often fails in patients with comorbidities. Tissue engineering combining functional cells, biomaterials and osteoinductive cues may provide alternative treatment strategies. We have recently demonstrated that stromal cell‐derived factor‐1β (SDF‐1β) works in concert with bone morphogenetic protein‐2 (BMP‐2) to potentiate osteogenic differentiation of bone marrow‐derived mesenchymal stem/stromal cells (BMSCs). Here, we test the hypothesis that SDF‐1β overexpressed in Tet‐Off‐SDF‐1β BMSCs, delivered on acellular dermal matrix (ADM), synergistically augments BMP‐2‐induced healing of critical‐sized mouse calvarial defects. BMSC therapies alone showed limited bone healing, which was increased with co‐delivery of BMP‐2. This was further enhanced in Tet‐Off‐SDF‐1β BMSCs + BMP‐2. Only limited BMSC retention on ADM constructs was observed after 4 weeks in vivo, which was increased with BMP‐2 co‐delivery. In vitro cell proliferation studies showed that supplementing BMP‐2 to Tet‐Off BMSCs significantly increased the cell number during the first 24 h. Consequently, the increased cell numbers decreased the detectable BMP‐2 levels in the medium, but increased cell‐associated BMP‐2. The data suggest that SDF‐1β provides synergistic effects supporting BMP‐2‐induced, BMSC‐mediated bone formation and appears suitable for optimization of bone augmentation in combination therapy protocols. Copyright © 2015 John Wiley & Sons, Ltd.     

D‐ and L‐[123I]‐2‐I‐phenylalanine show a long tumour retention compared with D‐ and L‐[123I]‐2‐I‐tyrosine in R1M rhabdomyosarcoma tumour‐bearing Wag/Rij rats

The aim of this study was the comparison of the tumour uptake and the long‐term retention of [¹²³I]‐2‐I‐L ‐phenylalanine and [¹²³I]‐2‐I‐D ‐phenylalanine with those of [¹²³I]‐2‐I‐L ‐tyrosine and [¹²³I]‐2‐I‐D ‐tyrosine in R1M rhabdomyosarcoma tumour‐bearing rats. The biodistribution of the radioactivity as a function of time in R1M tumour‐bearing rats was measured by planar gamma camera imaging (dynamic and static). If dissection was applied, the activity in the tumours and tissues of interest was measured by gamma counting. [¹²³I]‐2‐iodo‐L ‐phenylalanine, [¹²³I]‐2‐iodo‐D ‐phenylalaine, [¹²³I]‐2‐I‐L ‐tyrosine showed a considerable tumour uptake reaching a maximum between 10 and 30 min. At 30 min p.i. the differential uptake ratio values of this uptake were, respectively, 2.1, 2.3, 2.5 and 1.7. The activity in the tumour was shown to be related to a tumour cell uptake and not to an increased blood pool activity. All the tracers showed a clearance from the blood to the bladder without renal retention. At longer times both L ‐ and D ‐ [¹²³I]‐2‐I‐tyrosine were cleared for a large part from the tumours and the body. [¹²³I]‐2‐I‐L ‐Phe and [¹²³I]‐2‐I‐D ‐Phe showed a considerable and equal retention in the tumours: as compared with 0.5 h, 91% at 24 h and 80% at 48 h. This was related to the longer retention of activity in the blood pool noticed for these compounds (81% at 24 h and 65% at 48 h). The tumour‐to‐background ratio increased with 25% at those longer times. At short times all the tracers were taken up to a considerable extent in the tumours. In the R1M‐bearing Wag/Rij rat model only [¹²³I]‐2‐I‐L ‐phenylalanine and [¹²³I]‐2‐I‐D ‐phenylalanine showed an especially high retention at long times without any significant difference between the enantiomers. Copyright © 2007 John Wiley & Sons, Ltd.     

Clinical application of 18F‐fluoro‐2‐deoxy‐D‐glucose positron emission tomography/computed tomography in breast cancer

Positron emission tomography (PET)/computed tomography (CT) is not suited for primary diagnostics of breast tumours and it cannot replace sentinel lymph node technique in determining metastases to the axilla. PET/CT has a high sensitivity and specificity regarding the detection of loco‐regional recurrence and metastases to mediastinal and internal mammary lymph nodes, as well as distant metastases. Whether the method can replace conventional methods, or be a supplement when this is non‐conclusive, remains unresolved. PET/CT cannot be recommended for routine follow‐up but is recommended in patients with suspected relapse when conventional imaging has given equivocal results. PET/CT can be applied to confirm isolated loco‐regional relapse or metastatic lesion detected by conventional imaging. PET/CT has a high sensitivity for detecting response to treatment, but a low specificity calls for cautions. Further investigations into the use of PET/CT to predict and monitor response are warranted, before this approach may find its way into a clinical setting. In the future, PET/CT will probably find increasing use in treatment planning and evaluation of patients with breast cancer.     

Impact of H‐aggregation on activatable MMP‐2‐specific probes for optical imaging

In order to target and image MMP‐2 activity using optical imaging, we developed a panel of new MMP‐2 probes based on Cy5 and QSY21 as fluorophore/quencher FRET partners, separated by various MMP‐2 specific peptide substrates. We compared these probes for their specificity against other MMPs, their rate of activation by MMP‐2 and their initial quenching. Copyright © 2012 John Wiley & Sons, Ltd.     

Antibacterial and β‐Lactamase Inhibitory Activity of Monocyclic β‐Lactams

Due to the widespread emergence of resistant bacterial strains, an urgent need for the development of new antibacterial agents with novel modes of action has emerged. The discovery of naturally occurring monocyclic β‐lactams in the late 1970s, mainly active against aerobic Gram‐negative bacteria, has introduced a new approach in the design and development of novel antibacterial β‐lactam agents. The main goal was the derivatization of the azetidin‐2‐one core in order to improve their antibacterial potency, broaden their spectrum of activity, and enhance their β‐lactamase stability. In that respect, our review covers the updates in the field of monocyclic β‐lactam antibiotics during the last three decades, taking into account an extensive collection of references. An overview of the relationships between the structural features of these monocyclic β‐lactams, classified according to their N‐substituent, and the associated antibacterial or β‐lactamase inhibitory activities is provided. The different paragraphs disclose a number of well‐established classes of compounds, such as monobactams, monosulfactams, monocarbams, monophosphams, nocardicins, as well as other known representative classes. Moreover, this review draws attention to some less common but, nevertheless, possibly important types of monocyclic β‐lactams and concludes by highlighting the recent developments on siderophore‐conjugated classes of monocyclic β‐lactams.