In the present work, the methods of dynamic light scattering and fluorescence spectroscopy were applied to study the optical properties of aqueous dilutions of the humic substances complex (HC) as a potential drug delivery system. The supramolecular structures in the humate solution were characterized as monodisperse systems of the submicron range with a tendency to decrease in particle size with a decrease in the dry matter concentration. The slightly alkaline medium (8.3) of the studied aqueous dilutions of HC causes the absence of a pronounced fluorescence maximum in the region from 400 to 500 nm. However, the presence of an analytically significant, inversely proportional to the concentration second-order scattering (SOS) signal at 2λ_ex = λ_em was shown. In the examples of the antiviral substances mangiferin and favipiravir, it was shown that the use of the humic complex as a drug carrier makes it possible to increase the solubility by several times and simultaneously obtain a system with a smaller particle size of the dispersed phase. It has been shown that HC can interact with mangiferin and favipiravir to form stable structures, which lead to a significant decrease in SOS intensities on HC SOS spectra. The scattering wavelengths, λ_ex/λ_em, were registered at 350 nm/750 nm for mangiferin and 365 nm/730 nm for favipiravir, respectively. The increments of the scattering intensities (I0/I) turned out to be proportional to the concentration of antiviral components in a certain range of concentrations. Full article

Microneedles are transdermal drug delivery tools that can be fabricated simply, economically, and rapidly using SLA 3D printing. However, SLA 3D printing has a limitation in that the resolution is slightly lowered when the microneedle is precisely printed. To solve this issue, we optimized the SLA 3D printing conditions such as printing angle, needle height, aspect ratio, and spacing between the microneedles for high-resolution microneedle fabrication. The sharpest microneedle tip was obtained when the printing angle was adjusted to 60° in both the x and y axes. The aspect ratio and the spacing between the microneedles did not affect the output of the sharp tip. Under optimal conditions, the microneedles with 1180 ± 20 µm height, 490 ± 20 µm base, and 30.2 ± 3.4 µm tip diameter were obtained. The dissolving microneedle patch, prepared using the 3D printed microneedle as a mold, penetrated the porcine skin ex vivo. When the printing angle was 60° in the x and y axes, the area of the single stacking layer, including the microneedle tip, increased, and thus the sharp tip could be printed. A high-dimensional, side-notched arrowhead (SNA) microneedle was fabricated by applying the SLA 3D printing condition. Moreover, a letter-type microneedle patch was fabricated using the customized characteristics of 3D printing. Consequently, high-resolution and high-dimensional microneedles were successfully fabricated by adjusting the printing angle using a general SLA 3D printer, and this technology will be applied to the manufacture of drug delivery tools and various microstructures. Full article

Hot melt extrusion (HME), a continuous manufacturing process for generating supersaturating amorphous self-micellizing solid dispersion systems (saSMSDs), holds promise for achieving amorphization of many pharmaceutical formulations. For saSMSDs generation, HME-triggered continuous processes offer advantages over traditional non-continuous processes such as fusion/quench cooling (FQC) and co-precipitation (CP). Here we employed HME, FQC, and CP to generate saSMSDs containing the water-insoluble BCS II drug nitrendipine (NIT) and self-micellizing polymer Soluplus^®. Scanning electron microscopy, powder X-ray diffraction, and differential scanning calorimetry results revealed that saSMSDs formed when NIT–Soluplus^® mixtures were subjected to the abovementioned amorphization methods. All saSMSDs outperformed crystalline NIT preparations and physical mixtures in achieving extended supersaturable immediate release states with superior solubility, “spring-parachute” process characteristics, and dissolution behaviors. Notably, Fourier transform-infrared spectroscopic results obtained for saSMSDs detected hydrogen bonding interactions between the drug and the carrier. Ultimately, our results revealed the advantages of HME-triggered amorphization as a continuous process for significantly improving drug dissolution, increasing solubility, and maintaining supersaturation as compared to traditional amorphization-based techniques. Full article

Adenosine triphosphate (ATP)–competitive p97 inhibitor CB-5339, the successor of CB-5083, is being evaluated in Phase 1 clinical trials for anti-cancer therapy. Different modes-of-action p97 inhibitors such as allosteric inhibitors are useful to overcome drug-induced resistance, one of the major problems of targeted therapy. We previously demonstrated that allosteric p97 inhibitor NMS-873 can overcome CB-5083-induced resistance in HCT116. Here we employed chemical proteomics and drug-induced thermal proteome changes to identify drug targets, in combination with drug-resistant cell lines to dissect on- and off-target effects. We found that NMS-873 but not CB-5083 affected glycometabolism. By establishing NMS-873-resistant HCT116 cell lines and performing both cell-based and proteomic analysis, we confirmed that NMS-873 dysregulates glycometabolism in a p97-independent manner. We then used proteome integral solubility alteration with a temperature-based method (PISA T) to identify NDUFAF5 as one of the potential targets of NMS-873 in the mitochondrial complex I. We also demonstrated that glycolysis inhibitor 2-DG enhanced the anti-proliferative effect of NMS-873. The polypharmacology of NMS-873 can be advantageous for anti-cancer therapy for colon cancer. Full article

The present work presents translational research with application of AgNPs targeting the global drug resistance problem. In vivo fieldwork was carried out with 400 breeding farm cows sick with a serous mastitis. Ex vivo results revealed that after cow treatment with Lactobay^TM (a mixture of antibiotic drugs) the susceptibility to 31 antibiotics of S. aureus isolates from cow breast secretion decreased by 25%, while after treatment with Argovit–C^TM silver nanoparticles S. aureus susceptibility increased by 11%. The portion of isolates with an efflux effect leading to elimination of antibiotics from S. aureus after Lactobay-treatment resulted in a 15% increase, while Argovit-C-treatment led to a 17.5% decrease. The obtained results showed that mastitis treatments with Argovit-C^TM AgNPs can partially restore the activity of antibiotics towards S. aureus and shorten the duration of mastitis treatment by 33%. Full article

Atrial fibrillation (AF) is an arrhythmia associated with an increased stroke risk and mortality rate. Current treatment options leave unmet needs in AF therapy. Recently, doxapram has been introduced as a possible new option for AF treatment in a porcine animal model. To better understand its pharmacokinetics, three German Landrace pigs were treated with intravenous doxapram (1 mg/kg). Plasma and brain tissue samples were collected. For the analysis of these samples, an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the simultaneous measurement of doxapram and its active metabolite 2-ketodoxapram was developed and validated. The assay had a lower limit of quantification (LLOQ) of 10 pg/mL for plasma and 1 pg/sample for brain tissue. In pigs, doxapram pharmacokinetics were biphasic with a terminal elimination half-life (t_1/2) of 1.38 ± 0.22 h and a maximal plasma concentration (c_max) of 1780 ± 275 ng/mL. Its active metabolite 2-ketodoxapram had a t_1/2 of 2.42 ± 0.04 h and c_max of 32.3 ± 5.5 h after administration of doxapram. Protein binding was 95.5 ± 0.9% for doxapram and 98.4 ± 0.3% for 2-ketodoxapram with a brain-to-plasma ratio of 0.58 ± 0.24 for doxapram and 0.12 ± 0.02 for 2-ketodoxapram. In conclusion, the developed assay was successfully applied to the creation of pharmacokinetic data for doxapram, possibly improving the safety of its usage. Full article

Sufficient boron-10 isotope (¹⁰B) accumulation by tumor cells is one of the main requirements for successful boron neutron capture therapy (BNCT). The inability of the clinically registered ¹⁰B-containing borophenylalanine (BPA) to maintain a high boron tumor concentration during neutron irradiation after a single injection has been partially solved by its continuous infusion; however, its lack of persistence has driven the development of new compounds that overcome the imperfections of BPA. We propose using elemental boron nanoparticles (eBNPs) synthesized by cascade ultrasonic dispersion and destruction of elemental boron microparticles and stabilized with hydroxyethylcellulose (HEC) as a core component of a novel boron drug for BNCT. These HEC particles are stable in aqueous media and show no apparent influence on U251, U87, and T98G human glioma cell proliferation without neutron beam irradiation. In BNCT experiments, cells incubated with eBNPs or BPA at an equivalent concentration of 40 µg ¹⁰B/mL for 24 h or control cells without boron were irradiated at an accelerator-based neutron source with a total fluence of thermal and epithermal neutrons of 2.685, 5.370, or 8.055 × 10¹²/cm². The eBNPs significantly reduced colony-forming capacity in all studied cells during BNCT compared to BPA, verified by cell-survival curves fit to the linear-quadratic model and calculated radiobiological parameters, though the effect of both compounds differed depending on the cell line. The results of our study warrant further tumor targeting-oriented modifications of synthesized nanoparticles and subsequent in vivo BNCT experiments. Full article

Dendrimer–protein conjugates have significant prospects for biological applications. The complexation changes the biophysical behavior of both proteins and dendrimers. The dendrimers could influence the secondary structure of proteins, zeta-potential, distribution of charged regions on the surface, the protein–protein interactions, etc. These changes offer significant possibilities for the application of these features in nanotheranostics and biomedicine. Based on the dendrimer–protein interactions, several therapeutic applications of dendrimers have emerged. Thus, the formation of stable complexes retains the disordered proteins on the aggregation, which is especially important in neurodegenerative diseases. To clarify the origin of these properties and assess the efficiency of action, the mechanism of protein–dendrimer interaction and the nature and driving force of binding are considered in this review. The review outlines the antiamyloid activity of dendrimers and discusses the effect of dendrimer structures and external factors on their antiamyloid properties. Full article

Human lung deposition data is non-mandatory for drug approval but very useful for the development of orally inhaled drug products. Lung deposition of inhaled drugs can be quantified by radionuclide imaging, for which one of the first considerations is the method used to radiolabel formulations. In this study, we report the development of a radiolabeling method for lyophilizate for dry powder inhalation (LDPI) formulations. TechneCoat^TM is one method that can radiolabel drug particles without using solvents. In this method, particles are radiolabeled with a dispersion of ^99mTc-labeled nanoparticles called Technegas^TM. Because a LDPI formulation is not comprised of particles but is a lyophilized cake aerosolized by air impact, the TechneCoat method cannot be used for the radiolabeling of LDPI formulations. We therefore modified the TechneCoat apparatus so that LDPI formulations were not aerosolized by the Technegas flow. Radiolabeling using a modified TechneCoat apparatus was validated with model LDPI formulations of interferon alpha (IFN). IFN of ^99mTc-unlabeled, IFN of ^99mTc-labeled, and ^99mTc of ^99mTc-labeled LDPI formulations showed similar behavior, and differences from IFN of ^99mTc-unlabeled LDPI formulations were within ±15% in aerodynamic particle size distribution measurement. Our radiolabeling method for LDPI formulations may be useful for the quantification of drug deposition in human lungs. Full article

Acute myocardial infarction is a major global health problem, and the repair of damaged myocardium is still a major challenge. Myocardial injury triggers an inflammatory response: immune cells infiltrate into the myocardium while activating myofibroblasts and vascular endothelial cells, promoting tissue repair and scar formation. Fragments released by cardiomyocytes become endogenous “danger signals”, which are recognized by cardiac pattern recognition receptors, activate resident cardiac immune cells, release thrombin factors and inflammatory mediators, and trigger severe inflammatory responses. Inflammatory signaling plays an important role in the dilation and fibrosis remodeling of the infarcted heart, and is a key event driving the pathogenesis of post-infarct heart failure. At present, there is no effective way to reverse the inflammatory microenvironment in injured myocardium, so it is urgent to find new therapeutic and diagnostic strategies. Nanomedicine, the application of nanoparticles for the prevention, treatment, and imaging of disease, has produced a number of promising applications. This review discusses the treatment and challenges of myocardial injury and describes the advantages of functional nanoparticles in regulating the myocardial inflammatory microenvironment and overcoming side effects. In addition, the role of inflammatory signals in regulating the repair and remodeling of infarcted hearts is discussed, and specific therapeutic targets are identified to provide new therapeutic ideas for the treatment of myocardial injury. Full article

Together with the nucleus, the mitochondrion has its own genome. Mutations in mitochondrial DNA are responsible for a variety of disorders, including neurodegenerative diseases and cancer. Current therapeutic approaches are not effective. In this sense, mitochondrial gene therapy emerges as a valuable and promising therapeutic tool. To accomplish this goal, the design/development of a mitochondrial-specific gene delivery system is imperative. In this work, we explored the ability of novel polymer- and peptide-based systems for mitochondrial targeting, gene delivery, and protein expression, performing a comparison between them to reveal the most adequate system for mitochondrial gene therapy. Therefore, we synthesized a novel mitochondria-targeting polymer (polyethylenimine–dequalinium) to load and complex a mitochondrial-gene-based plasmid. The polymeric complexes exhibited physicochemical properties and cytotoxic profiles dependent on the nitrogen-to-phosphate-group ratio (N/P). A fluorescence confocal microscopy study revealed the mitochondrial targeting specificity of polymeric complexes. Moreover, transfection mediated by polymer and peptide delivery systems led to gene expression in mitochondria. Additionally, the mitochondrial protein was produced. A comparative study between polymeric and peptide/plasmid DNA complexes showed the great capacity of peptides to complex pDNA at lower N/P ratios, forming smaller particles bearing a positive charge, with repercussions on their capacity for cellular transfection, mitochondria targeting and, ultimately, gene delivery and protein expression. This report is a significant contribution to the implementation of mitochondrial gene therapy, instigating further research on the development of peptide-based delivery systems towards clinical translation. Full article

Cerium oxide nanoparticles (CNPs), owing to their antioxidant property, have recently emerged as therapeutic candidate for Alzheimer’s disease (AD). However, intravenous CNPs are limited due to their poor physicochemical properties, rapid blood clearance and poor blood–brain penetration. Thus, we developed intranasal CNPs and evaluated its potential in experimental AD. CNPs were synthesized using homogenous precipitation method and optimized through Box–Behnken Design. The formation of CNPs was confirmed by UV spectroscopy and FTIR. The optimized CNP were spherical, small (134.0 ± 3.35 nm), uniform (PDI, 0.158 ± 0.0019) and stable (ZP, −21.8 ± 4.94 mV). The presence of Ce in CNPs was confirmed by energy-dispersive X-ray analysis. Further, the X-ray diffraction spectra revealed that the CNPs were nano-crystalline. The DPPH assay showed that at concentration of 50 µg/mL, the percentage radical scavenging was 95.40 ± 0.006%. Results of the in vivo behavioral studies in the scopolamine-induced Alzheimer rat model showed that intranasal CNPs dose dependently reversed cognitive ability. At dose of 6 mg/kg the morris water maze results (escape latency, path length and dwell time) and passive avoidance results (retention latency) were significantly different from untreated group but not significantly different from positive control group (rivastigmine patch, 13.3 mg/24 h). Further, biochemical estimation showed that intranasal CNP upregulated the levels of SOD and GSH in brain. In conclusion, intranasal CNPs, through its antioxidant effect, could be a prospective therapeutics for the treatment of cognitive impairment in AD. Full article

Gastric ulcer is one of the most frequent gastrointestinal disorders, and there is an increasing search for natural products that can heal ulcers and avoid their recurrence. We aimed to evaluate the gastroprotective activity of vanillin, including the investigation of anti-inflammatory activity and the modulation of gene expression. Wistar rats were orally treated with vehicle, carbenoxolone, or vanillin (25, 50, or 100 mg/kg) and orally received absolute ethanol to develop gastric ulcers. We analyzed the ulcer area, conducted histological analysis, and measured the levels of the inflammatory cytokines TNF-α, IL-6, IL-1β, and IFN-γ, and anti-inflammatory cytokine IL-10 by ELISA. We analyzed mRNA expression for NF-κB, TNF-α, and Il-10. We measured NOx levels using the Griess reaction. Our results showed similar gastroprotection for the three doses. Vanillin increased mucus production and preserved gastric mucosa integrity. The gastroprotective effect was linked to anti-inflammatory activity as a result of decreasing the levels of TNF-α, IL-6, IL-1β, and IFN-γ and increasing IL-10 levels. Vanillin downregulated the mRNA expression of NF-κB and TNF-α, upregulated the mRNA expression of Il-10, and increased NOx levels in the stomach. The gastroprotective activity of vanillin is related to the maintenance of gastric mucus and the local inflammatory response modulation. Full article

As a widely distributed parasitic nematode of ruminants, Haemonchus contortus has become resistant to most anthelmintic classes, there has been a major demand for new compounds against H. contortus and related nematodes. Recent phenotypic screening has revealed two compounds, designated as BLK127 and HBK4, that are active against H. contortus larvae. The present study was designed to assess the activity of these compounds against H. contortus eggs and adults, hepatotoxicity in rats and sheep, as well as biotransformation in H. contortus adults and the ovine liver. Both compounds exhibited no inhibitory effect on the hatching of eggs. The benzyloxy amide BLK127 significantly decreased the viability of adults in sensitive and resistant strains of H. contortus and showed no hepatotoxic effect, even at the highest concentration tested (100 µM). In contrast, HBK4 had no impact on the viability of H. contortus adults and exhibited significant hepatotoxicity. Based on these findings, HBK4 was excluded from further studies, while BLK127 seems to be a potential candidate for a new anthelmintic. Consequently, biotransformation of BLK127 was tested in H. contortus adults and the ovine liver. In H. contortus, several metabolites formed via hydroxylation, hydrolysis and glycosidation were identified, but the extent of biotransformation was low, and the total quantity of the metabolites formed did not differ significantly between the sensitive and resistant strains. In contrast, ovine liver cells metabolized BLK127 more extensively with a glycine conjugate of 4-(pentyloxy)benzoic acid as the main BLK127 metabolite. Full article

Linezolid is an efficacious medication for the treatment of drug-resistant tuberculosis but has been associated with serious safety issues that can result in treatment interruption. The objectives of this study were thus to build a population pharmacokinetic model and to use the developed model to establish a model-informed precision dosing (MIPD) algorithm enabling safe and efficacious dosing in patients with multidrug- and extensively drug-resistant tuberculosis. Routine hospital therapeutic drug monitoring data, collected from 70 tuberculosis patients receiving linezolid, was used for model development. Efficacy and safety targets for MIPD were the ratio of unbound area under the concentration versus time curve between 0 and 24 h over minimal inhibitory concentration (fAUC_0–24h/MIC) above 119 and unbound plasma trough concentration (fC_min) below 1.38 mg/L, respectively. Model building was performed in NONMEM 7.4.3. The final population pharmacokinetic model consisted of a one-compartment model with transit absorption and concentration- and time-dependent auto-inhibition of elimination. A flat dose of 600 mg once daily was appropriate in 67.2% of the simulated patients from an efficacy and safety perspective. Using the here developed MIPD algorithm, the proportion of patients reaching the efficacy and safety target increased to 81.5% and 88.2% using information from two and three pharmacokinetic sampling occasions, respectively. This work proposes an MIPD approach for linezolid and suggests using three sampling occasions to derive an individualized dose that results in adequate efficacy and fewer safety concerns compared to flat dosing. Full article