Pharmaceuticals — Open Access Journal

The demand for reliable comparability studies of biosimilars grows with their increased market share. These studies focus on physicochemical, structural, functional and clinical properties to ensure that a biosimilar has no significant differences to the originator product and can be released into the market without extensive clinical trials. In the current study, Enbrel^® (etanercept, the originator) and Altebrel™ (the proposed biosimilar) underwent direct comparison. “Bottom-up” mass spectrometric analysis was used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of N-glycans on in-vitro functionality of etanercept was assayed. Three enzyme peptide mapping resulted in complete identification of the primary structure. It was confirmed that total ion chromatograms are valuable datasets for the analysis of the primary structure of biodrugs. New N/O-glycan structures were identified and all the N-glycans were quantified. Finally, investigation of the functional properties of N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis and in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality. Analysis of etanercept and its biosimilar, revealed a high similarity in terms of glycosylation, primary structure and in-vitro functionality. Full article

The high overexpression of cholecystokinin-2 receptors (CCK2R) in tumors, such as medullary thyroid carcinoma, allows for highly specific diagnostic and therapeutic targeting with radiolabeled peptide probes derived from natural ligands for the receptor. Based on the ideal imaging characteristics, high availability and low cost of technetium-99m (^99mTc)-labeled radiopharmaceuticals we have developed two hydrazinonicotinic acid (HYNIC) conjugated minigastrin analogs allowing labeling at high specific activity. The CCK2R targeting peptide conjugates show specific amino acid substitutions in the C-terminal receptor-specific sequence with the aim to increase stability and tumor targeting. The CCK2R affinity and the cell uptake of the new radioligands were analyzed using A431 human epidermoid carcinoma cells stably transfected with human CCK2R and mock transfected cells. Metabolic studies in BALB/c mice revealed a high resistance against enzymatic degradation for both radioligands. Biodistribution studies in tumor-xenografted athymic BALB/c nude mice at 1 h and 4 h p.i. showed that the two ^99mTc-labeled compounds showed varying uptake in receptor expressing organs, stomach and pancreas (1.3–10.4% IA/g), as well as kidneys, the main route of excretion (7.8–19.9% IA/g). The tumor uptake in A431-CCK2R xenografts was 24.75 ± 4.38% IA/g for [^99mTc]Tc-HYNIC-MGS5 and 42.48 ± 6.99% IA/g for [^99mTc]Tc-HYNIC-MGS11 at 4 h p.i., whereas the tumor-to-kidney ratio was comparable (2.6–3.3). On demand availability and potential application for radioguided surgery of a ^99mTc-labeled minigastrin analog support the further evaluation of these highly promising new compounds. Full article

The tracer [[11C]meta-Hydroxyephedrine ([[11C]mHED) is one of the most applied PET tracers for cardiac imaging, whose radiosynthesis was already reported in 1990. While not stated in the literature, separation difficulties and an adequate formulation of the product are well known challenges in its production. Furthermore, the precursor (metaraminol) is also a substrate for the norepinephrine transporter, and can therefore affect the image quality. This study aims at optimizing the synthetic process of [[11C]mHED and investigating the effect of the apparent molar activity (sum of mHED and metaraminol) in patients and animals. The main optimization was the improved separation through reverse phase-HPLC by a step gradient and subsequent retention of the product on a weakly-cationic ion exchange cartridge. The µPET/µCT was conducted in ten rats (ischemic model) and the apparent molar activity was correlated to the VOI- and SUV-ratio of the myocardium/intra-ventricular blood pool. Moreover, nine long-term heart transplanted and five Morbus Fabry patients underwent PET and MRI imaging for detection of changes in the sympathetic innervation. In summary, the fully-automated synthesis and optimized purification method of [[11C]mHED is easily applicable and reproducible. Moreover, it was shown that the administered apparent molar activities had a negligible effect on the imaging quality. Full article

Naringenin is a flavonoid belonging to flavanones subclass. It is widely distributed in several Citrus fruits, bergamot, tomatoes and other fruits, being also found in its glycosides form (mainly naringin). Several biological activities have been ascribed to this phytochemical, among them antioxidant, antitumor, antiviral, antibacterial, anti-inflammatory, antiadipogenic and cardioprotective effects. Nonetheless, most of the data reported have been obtained from in vitro or in vivo studies. Although some clinical studies have also been performed, the main focus is on naringenin bioavailability and cardioprotective action. In addition, these studies were done in compromised patients (i.e., hypercholesterolemic and overweight), with a dosage ranging between 600 and 800 μM/day, whereas the effect on healthy volunteers is still debatable. In fact, naringenin ability to improve endothelial function has been well-established. Indeed, the currently available data are very promising, but further research on pharmacokinetic and pharmacodynamic aspects is encouraged to improve both available production and delivery methods and to achieve feasible naringenin-based clinical formulations. Full article

Juvenile idiopathic arthritis (JIA) represents joint inflammation with an unknown cause that starts before the age of 16, resulting in stiff and painful joints. In addition, JIA patients often report symptoms of sickness behavior. Recent animal studies suggest that proinflammatory cytokines produce sickness behavior by increasing the activity of indoleamine-2,3-dioxygenase (IDO) and guanosinetriphosphate–cyclohydrolase-1 (GTP–CH1). Here, it is hypothesized that inflammation in JIA patients enhances the enzymatic activity of IDO and GTP-CH1 and decreases the co-factor tetrahydrobiopterin (BH4). These compounds play a crucial role in the synthesis and metabolism of neurotransmitters. The aim of our study was to reveal whether inflammation affects both the GTP-CH1 and IDO pathway in JIA patients. Serum samples were collected from twenty-four JIA patients. In these samples, the concentrations of tryptophan (TRP), kynurenine (KYN), tyrosine (TYR), neopterin, and phenylalanine (PHE) were measured. An HPLC method with electrochemical detection was developed to quantify tryptophan, kynurenine, and tyrosine. Neopterin and phenylalanine were quantified by ELISA. The KYN/TRP ratio was measured as an index of IDO activity, while the PHE/TYR ratio was measured as an index of BH4 activity. Neopterin concentrations were used as an indirect measure of GTP-CH1 activity. JIA patients with high disease activity showed higher levels of both neopterin and kynurenine, and a higher ratio of both KYN/TRP and PHE/TYR and lower tryptophan levels than clinically inactive patients. Altogether, these data support our hypothesis that inflammation increases the enzymatic activity of both IDO and GTP-CH1 but decreases the efficacy of the co-factor BH4. In the future, animal studies are needed to investigate whether inflammation-induced changes in these enzymatic pathways and co-factor BH4 lower the levels of the brain neurotransmitters glutamate, noradrenaline, dopamine, serotonin, and melatonin, and consequently, whether they may affect fatigue, cognition, anxiety, and depression. Understanding of these complex neuroimmune interactions provides new possibilities for Pharma-Food interventions to improve the quality of life of patients suffering from chronic inflammation. Full article

The importance of Ca²⁺ signalling in key events of cancer cell function and tumour progression, such as proliferation, migration, invasion and survival, has recently begun to be appreciated. Many cellular Ca²⁺-stimulated signalling cascades utilise the intermediate, calmodulin (CaM). The Ca²⁺/CaM complex binds and activates a variety of enzymes, including members of the multifunctional Ca²⁺/calmodulin-stimulated protein kinase (CaMK) family. These enzymes control a broad range of cancer-related functions in a multitude of tumour types. Herein, we explore the cancer-related functions of these kinases and discuss their potential as targets for therapeutic intervention. Full article

The tropomyosin receptor kinases family (TrkA, TrkB, and TrkC) supports neuronal growth, survival, and differentiation during development, adult life, and aging. TrkA/B/C downregulation is a prominent hallmark of various neurological disorders including Alzheimer’s disease (AD). Abnormally expressed or overexpressed full-length or oncogenic fusion TrkA/B/C proteins were shown to drive tumorigenesis in a variety of neurogenic and non-neurogenic human cancers and are currently the focus of intensive clinical research. Neurologic and oncologic studies of the spatiotemporal alterations in TrkA/B/C expression and density and the determination of target engagement of emerging antineoplastic clinical inhibitors in normal and diseased tissue are crucially needed but have remained largely unexplored due to the lack of suitable non-invasive probes. Here, we review the recent development of carbon-11- and fluorine-18-labeled positron emission tomography (PET) radioligands based on specifically designed small molecule kinase catalytic domain-binding inhibitors of TrkA/B/C. Basic developments in medicinal chemistry, radiolabeling and translational PET imaging in multiple species including humans are highlighted. Full article

Aptamers were first described almost 30 years ago, with the publication of three separate research papers describing how a randomized library of RNA sequences could be incubated with a target to find a sequence that specifically binds via van der Waals forces, covalent and hydrogen bonding, and not Watson Crick base pairing [...] Full article

A strong mechanistic link between the regulation of iron homeostasis and oxygen sensing is evident in the lung, where both systems must be properly controlled to maintain lung function. Imbalances in pulmonary iron homeostasis are frequently associated with respiratory diseases, such as chronic obstructive pulmonary disease and with lung cancer. However, the underlying mechanisms causing alterations in iron levels and the involvement of iron in the development of lung disorders are incompletely understood. Here, we review current knowledge about the regulation of pulmonary iron homeostasis, its functional importance, and the link between dysregulated iron levels and lung diseases. Gaining greater knowledge on how iron contributes to the pathogenesis of these diseases holds promise for future iron-related therapeutic strategies. Full article

The need for new drugs is compelling, irrespective of the disease. Focusing on medical problems in the Western countries, heart disease and cancer are at the moment predominant illnesses. Owing to the fact that ~90% of all 21,000 cellular proteins in humans are regulated by phosphorylation/dephosphorylation it is not surprising that the enzymes catalysing these reactions (i.e., protein kinases and phosphatases, respectively) have attracted considerable attention in the recent past. Protein kinases are major team players in cell signalling. In tumours, these enzymes are found to be mutated disturbing the proper function of signalling pathways and leading to uncontrolled cellular growth and sustained malignant behaviour. Hence, the search for small-molecule inhibitors targeting the altered protein kinase molecules in tumour cells has become a major research focus in the academia and pharmaceutical companies. Full article

Introduction: Benzotriazine-1,4-dioxides (BTDOs) such as tirapazamine (TPZ) and its derivatives act as radiosensitizers of hypoxic tissues. The benzotriazine-1-monoxide (BTMO) metabolite (SR 4317, TPZMO) of TPZ also has radiosensitizing properties, and via unknown mechanisms, is a potent enhancer of the radiosensitizing effects of TPZ. Unlike their 2-nitroimidazole radiosensitizer counterparts, radiolabeled benzotriazine oxides have not been used as radiopharmaceuticals for diagnostic imaging or molecular radiotherapy (MRT) of hypoxia. The radioiodination chemistry for preparing model radioiodinated BTDOs and BTMOs is now reported. Hypothesis: Radioiodinated 3-(2-iodoethoxyethyl)-amino-1,2,4-benzotriazine-1,4-dioxide (I-EOE-TPZ), a novel bioisosteric analogue of TPZ, and 3-(2-iodoethoxyethyl)-amino-1,2,4-benzotriazine-1-oxide (I-EOE-TPZMO), its monoxide analogue, are candidates for in vivo and in vitro investigations of biochemical mechanisms in pathologies that develop hypoxic microenvironments. In theory, both radiotracers can be prepared from the same precursors. Methods: Radioiodination procedures were based on classical nucleophilic [¹³¹I]iodide substitution on Tos-EOE-TPZ (P1) and by [¹³¹I]iodide exchange on I-EOE-TPZ (P2). Reaction parameters, including temperature, reaction time, solvent and the influence of pivalic acid on products’ formation and the corresponding radiochemical yields (RCY) were investigated. Results: The [¹³¹I]iodide labeling reactions invariably led to the synthesis of both products, but with careful manipulation of conditions the preferred product could be recovered as the major product. Radioiodide exchange on P2 in ACN at 80 ± 5 °C for 30 min afforded the highest RCY, 89%, of [¹³¹I]I-EOE-TPZ, which upon solid phase purification on an alumina cartridge gave 60% yield of the product with over 97% of radiochemical purity. Similarly, radioiodide exchange on P2 in ACN at 50 ± 5 °C for 30 min with pivalic acid afforded the highest yield, 92%, of [¹³¹I]I-EOE-TPZMO exclusively with no trace of [¹³¹I]I-EOE-TPZ. In both cases, extended reaction times and/or elevated temperatures resulted in the formation of at least two additional radioactive reaction products. Conclusions: Radioiodination of P1 and P2 with [¹³¹I]iodide leads to the facile formation of [¹³¹I]I-EOE-TPZMO. At 80 °C and short reaction times, the facile reduction of the N-4-oxide moiety was minimized to afford acceptable radiochemical yields of [¹³¹I]I-EOE-TPZ from either precursor. Regeneration of [¹³¹I]I-EOE-TPZ from [¹³¹I]I-EOE-TPZMO is impractical after reaction work-up. Full article

Cancer has a high incidence and mortality rate worldwide, which continues to grow as millions of people are diagnosed annually. Metastatic disease caused by cancer is largely responsible for the mortality rates, thus early detection of metastatic tumours can improve prognosis. However, a large number of patients will also present with micrometastasis tumours which are often missed, as conventional medical imaging modalities are unable to detect micrometastases due to the lack of specificity and sensitivity. Recent advances in radiochemistry and the development of nucleic acid based targeting molecules, have led to the development of novel agents for use in cancer diagnostics. Monoclonal antibodies may also be used, however, they have inherent issues, such as toxicity, cost, unspecified binding and their clinical use can be controversial. Aptamers are a class of single-stranded RNA or DNA ligands with high specificity, binding affinity and selectivity for a target, which makes them promising for molecular biomarker imaging. Aptamers are presented as being a superior choice over antibodies because of high binding affinity and pH stability, amongst other factors. A number of aptamers directed to cancer cell markers (breast, lung, colon, glioblastoma, melanoma) have been radiolabelled and characterised to date. Further work is ongoing to develop these for clinical applications. Full article

Currently, one of the greatest health challenges worldwide is the resistance to antibiotic drugs, which has led to the pursuit of new alternatives for the recovery of biological activity, where the use of different types of nano-systems has shown an interesting potential. In this study, we evaluated the antibiotic activity of a model drug (ampicillin) encapsulated within coated-nanoliposomes on strains of Staphylococcus aureus with different antibiotic-resistance degrees. Hence, liposomes were elaborated by the ethanol injection method and were coated with a cationic polymer (Eudragit E-100) through the layer-by-layer process. Liposome characterization, such as size, polydispersity, zeta potential, and encapsulation efficiency were determined using dynamic light scattering and ultrafiltration/centrifugation techniques. Although biological activity was evaluated using three ATCC strains of S. aureus corresponding to ATCC 25923 (sensitive), ATCC 29213 (resistant) and ATCC 43300 (very resistant). The results showed changes in size (from ~150 to 220 nm), polydispersity (from 0.20 to 0.45) and zeta potential (from −37 to +45 mV) for the coating process. In contrast, encapsulation efficiency of approximately 70% and an increase in antibiotic activity of 4 and 18 times more on those S. aureus-resistant strains have been observed. Full article

Differential scanning calorimetry (DSC) has emerged as a helpful technique both to characterize drug delivery systems and to study their interactions with bio-membranes. In this work, we compared idebenone (IDE)-loaded solid lipid nanoparticle (SLN) interactions with bio-membranes assessed by DSC with previous in vitro skin penetration data to evaluate the feasibility of predicting IDE skin penetration using DSC analyses. In vitro interactions experiments were performed using multi-lamellar liposomes as a model of bio-membrane. Enthalpy changes (ΔH) and transition temperature (Tm) were assessed during nine repeated DSC scans to evaluate IDE-loaded SLN–bio-membrane interactions over time. Analyzing ΔH and Tm values for each scan, we observed that the difference of ΔH and Tm values between the first and the last scan seemed to be related to SLN ability to locate IDE in the epidermis and in the stratum corneum, respectively. Therefore, the results of this study suggest the possibility of qualitatively predicting in vitro IDE skin penetration from IDE-loaded SLN utilizing the calorimetric parameters obtained from interaction experiments between the carriers under investigation and a model of bio-membrane. Full article

Macrophages are sentinel cells of the innate immune system and have important functions in development, tissue homeostasis, and immunity. These phylogenetically ancient cells also developed a variety of mechanisms to control erythropoiesis and the handling of iron. Red pulp macrophages in the spleen, Kupffer cells in the liver, and central nurse macrophages in the bone marrow ensure a coordinated metabolism of iron to support erythropoiesis. Phagocytosis of senescent red blood cells by macrophages in the spleen and the liver provide a continuous delivery of recycled iron under steady-state conditions and during anemic stress. Central nurse macrophages in the bone marrow utilize this iron and provide a cellular scaffold and niche to promote differentiation of erythroblasts. This review focuses on the role of the distinct macrophage populations that contribute to efficient iron metabolism and highlight important cellular and systemic mechanisms involved in iron-regulating processes. Full article

Positron emission tomography (PET) imaging of Colony Stimulating Factor 1 Receptor (CSF1R) is a new strategy for quantifying both neuroinflammation and inflammation in the periphery since CSF1R is expressed on microglia and macrophages. AZ683 has high affinity for CSF1R (K_i = 8 nM; IC₅₀ = 6 nM) and >250-fold selectivity over 95 other kinases. In this paper, we report the radiosynthesis of [¹¹C]AZ683 and initial evaluation of its use in CSF1R PET. [¹¹C]AZ683 was synthesized by ¹¹C-methylation of the desmethyl precursor with [¹¹C]MeOTf in 3.0% non-corrected activity yield (based upon [¹¹C]MeOTf), >99% radiochemical purity and high molar activity. Preliminary PET imaging with [¹¹C]AZ683 revealed low brain uptake in rodents and nonhuman primates, suggesting that imaging neuroinflammation could be challenging but that the radiopharmaceutical could still be useful for peripheral imaging of inflammation. Full article

Inflammation, being a hallmark of many chronic diseases, including cancer, inflammatory bowel disease, rheumatoid arthritis, and chronic kidney disease, negatively affects iron homeostasis, leading to iron retention in macrophages of the mononuclear phagocyte system. Functional iron deficiency is the consequence, leading to anemia of inflammation (AI). Iron deficiency, regardless of anemia, has a detrimental impact on quality of life so that treatment is warranted. Therapeutic strategies include (1) resolution of the underlying disease, (2) iron supplementation, and (3) iron redistribution strategies. Deeper insights into the pathophysiology of AI has led to the development of new therapeutics targeting inflammatory cytokines and the introduction of new iron formulations. Moreover, the discovery that the hormone, hepcidin, plays a key regulatory role in AI has stimulated the development of several therapeutic approaches targeting the function of this peptide. Hence, inflammation-driven hepcidin elevation causes iron retention in cells and tissues. Besides pathophysiological concepts and diagnostic approaches for AI, this review discusses current guidelines for iron replacement therapies with special emphasis on benefits, limitations, and unresolved questions concerning oral versus parenteral iron supplementation in chronic inflammatory diseases. Furthermore, the review explores how therapies aiming at curing the disease underlying AI can also affect anemia and discusses emerging hepcidin antagonizing drugs, which are currently under preclinical or clinical investigation. Full article

Permeation models are often used to determine diffusion properties of a drug through a membrane as it is released from a delivery system. In order to circumvent problematic in vivo studies, diffusion studies can be performed in vitro, using (semi-)synthetic membranes. In this study salicylic acid permeation was studied, employing a nitrocellulose membrane. Both saturated and unsaturated salicylic acid solutions were studied. Additionally, the transport of salicylic acid through the nitrocellulose membrane was simulated by computational modelling. Experimental observations could be explained by the transport mechanism that was revealed by dissipative particle dynamics (DPD) simulations. The DPD model was developed with the aid of atomistic scale molecular dynamics (AA-MD). The choice of a suitable model membrane can therefore, be predicted by AA-MD and DPD simulations. Additionally, the difference in the magnitude of release from saturated and unsaturated salicylic acid and solutions could also be observed with DPD. Moreover, computational studies can reveal hidden variables such as membrane-permeant interaction that cannot be measured experimentally. A recommendation is made for the development of future model permeation membranes is to incorporate computational modelling to aid the choice of model. Full article