Suramin: Effectiveness of analogues reveals structural features that are important for the potent trypanocidal activity of the drug.

Suramin was the first effective drug for the treatment of human African sleeping sickness. Structural analogues of the trypanocide have previously been shown to be potent inhibitors of several enzymes. Therefore, four suramin analogues lacking the methyl group on the intermediate rings and with different regiochemistry of the naphthalenetrisulphonic acid groups and the phenyl rings were tested to establish whether they exhibited improved antiproliferative activity against bloodstream forms of Trypanosomes brucei compared to the parent compound. The four analogues exhibited low trypanocidal activity and weak inhibition of the antitrypanosomal activity of suramin in competition experiments. This indicates that the strong trypanocidal activity of suramin is most likely due to the presence of methyl groups on its intermediate rings and to the specific regiochemistry of naphthalenetrisulphonic acid groups. These two structural features are also likely to be important for the inhibition mechanism of suramin because DNA distribution and nucleus/kinetoplast configuration analyses suggest that the analogues inhibit mitosis while suramin inhibits cytokinesis.

Suramin analogues protect cartilage against osteoarthritic breakdown by increasing levels of tissue inhibitor of metalloproteinases 3 (TIMP-3) in the tissue.

Osteoarthritis is a chronic degenerative joint disease affecting millions of people worldwide, with no disease-modifying drugs currently available to treat the disease. Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a potential therapeutic target in osteoarthritis because of its ability to inhibit the catabolic metalloproteinases that drive joint damage by degrading the cartilage extracellular matrix. We previously found that suramin inhibits cartilage degradation through its ability to block endocytosis and intracellular degradation of TIMP-3 by low-density lipoprotein receptor-related protein 1 (LRP1), and analysis of commercially available suramin analogues indicated the importance of the 1,3,5-trisulfonic acid substitutions on the terminal naphthalene rings for this activity. Here we describe synthesis and structure-activity relationship analysis of additional suramin analogues using ex vivo models of TIMP-3 trafficking and cartilage degradation. This showed that 1,3,6-trisulfonic acid substitution of the terminal naphthalene rings was also effective, and that the protective activity of suramin analogues depended on the presence of a rigid phenyl-containing central region, with para/para substitution of these phenyl rings being most favourable. Truncated analogues lost protective activity. The physicochemical characteristics of suramin and its analogues indicate that approaches such as intra-articular injection would be required to develop them for therapeutic use.

Fructose malabsorption: causes, diagnosis and treatment.

This review intends to act as an overview of fructose malabsorption (FM) and its role in the aetiology of diseases including, but not limited to, irritable bowel syndrome (IBS) and infantile colic and the relationship between fructose absorption and the propagation of some cancers. IBS results in a variety of symptoms including stomach pains, cramps and bloating. Patients can be categorised into two groups, depending on whether the patients' experiences either constipation (IBS-C) or diarrhoea (IBS-D). FM has been proposed as a potential cause of IBS-D and other diseases, such as infantile colic. However, our knowledge of FM is limited by our understanding of the biochemistry related to the absorption of fructose in the small intestine and FM's relationship with small intestinal bacterial overgrowth. It is important to consider the dietary effects on FM and most importantly, the quantity of excess free fructose consumed. The diagnosis of FM is difficult and often requires indirect means that may result in false positives. Current treatments of FM include dietary intervention, such as low fermentable oligo-, di-, monosaccharides and polyols diets and enzymatic treatments, such as the use of xylose isomerase. More research is needed to accurately diagnose and effectively treat FM. This review is designed with the goal of providing a detailed outline of the issues regarding the causes, diagnosis and treatment of FM.

Monitoring apoptosis in intact cells by high-resolution magic angle spinning 1 H NMR spectroscopy.

Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in 1 H NMR-observable resonances associated with lipids stored in lipid droplets. However, standard one-dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis-based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution-state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS 1 H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1 H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR-based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with 1 H NMR spectroscopy-derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.

Enzyme inhibition as a potential therapeutic strategy to treat COVID-19 infection.

With the emergence of the third infectious and virulent coronavirus within the past two decades, it has become increasingly important to understand how the virus causes infection. This will inform therapeutic strategies that target vulnerabilities in the vital processes through which the virus enters cells. This review identifies enzymes responsible for SARS-CoV-2 viral entry into cells (ACE2, Furin, TMPRSS2) and discuss compounds proposed to inhibit viral entry with the end goal of treating COVID-19 infection. We argue that TMPRSS2 inhibitors show the most promise in potentially treating COVID-19, in addition to being a pre-existing medication with fewer predicted side-effects.

NMR and Thermal Studies for the Characterization of Mass Transport and Phase Separation in Paracetamol/Copovidone Hot-Melt Extrusion Formulations.

The formulation of drug/polymer amorphous solid dispersions (ASDs) is one of the most successful strategies for improving the oral bioavailability of poorly soluble active pharmaceutical ingredients (APIs). Hot-melt extrusion (HME) is one method for preparing ASDs that is growing in importance in the pharmaceutical industry, but there are still substantial gaps in our understanding regarding the dynamics of drug dissolution and dispersion in viscous polymers and the physical stability of the final formulations. Furthermore, computational models have been built to predict optimal processing conditions, but they are limited by the lack of experimental data for key mass transport parameters, such as the diffusion coefficient. The work presented here reports direct measurements of API diffusion in pharmaceutical polymer melts, using high-temperature pulsed-field gradient NMR. The diffusion coefficient of a model drug/polymer system (paracetamol/copovidone) was determined for different drug loadings and at temperatures relevant to the HME process. The mechanisms of the diffusion process are also explored with the Stokes-Einstein and Arrhenius models. The results show that diffusivity is linked exponentially to temperature. Furthermore, this study includes rheological characterization, differential scanning calorimetry (DSC), and 1H ssNMR T1 and T1ρ measurements to give additional insights into the physical state, phase separation, and API/polymer interactions in paracetamol/copovidone ASD formulations.

Suramin Inhibits Osteoarthritic Cartilage Degradation by Increasing Extracellular Levels of Chondroprotective Tissue Inhibitor of Metalloproteinases 3.

Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3, through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor-related protein 1 (LRP1). We discovered that suramin (C51H40N6O23S6) bound to TIMP-3 with a KD value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin-like metalloproteinase with thrombospondin motifs 5. NF279 (8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt), a structural analog of suramin, has an increased affinity for TIMP-3 and increased ability to inhibit TIMP-3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis.

Indomethacin-Kollidon VA64 Extrudates: A Mechanistic Study of pH-Dependent Controlled Release.

Because of its weakly acidic nature (pKa of 4.5), indomethacin presents an aqueous solubility that significantly increases when changing from acidic to neutral/alkaline pH (1.5 µg/mL at pH 1.2 and 105.2 µg/mL at pH 7.4). We have therefore investigated the impact of the dissolution medium pH on the dissolution performance of indomethacin:Kollidon VA64 extrudates. The impact of the drug loading on the dissolution properties of these systems was also examined (5%, 15%, 30%, 50%, 70%, and 90% drug loading). Time-resolved Raman spectroscopy along with in-line UV-vis spectrophotometry was employed to directly relate changes in dissolution behavior to physicochemical changes that occur to the extrudate during the test. The dissolution tests were performed in pH 2 HCl (to mimic the stomach conditions), and this was then switched during the experiment to pH 6.8 phosphate buffer (to simulate the poststomach conditions). The rotating disc dissolution rate test was also used to simultaneously measure the dissolution rate of both the drug and the polymer. We found that in pH 2 HCl buffer, for the 15% or higher drug-loaded extrudates, Kollidon VA64 preferentially dissolves from the exterior of the compact leaving an amorphous drug-rich hydrophobic shell, which, similarly to an enteric coating, inhibits the drug release. The in situ formation of an enteric coating has been previously hypothesized, and this has been the first time that is directly observed in a pH-variable dissolution test. The dissolution medium switch to pH 6.8 phosphate buffer, due to the large increase of the aqueous solubility of indomethacin at this pH, leads to rapid dissolution of the material forming the coating and therefore total drug release. In contrast, the 5% extrudate is fully hydrated and quickly dissolves at low pH pointing to a dissolution performance dependent on highly water-soluble Kollidon VA64.

Monitoring the Dissolution Mechanisms of Amorphous Bicalutamide Solid Dispersions via Real-Time Raman Mapping.

Real-time in situ Raman mapping has been employed to monitor, during dissolution, the crystallization transitions of amorphous bicalutamide formulated as a molecular dispersion in a copovidone VA64 matrix. The dissolution performance was also investigated using the rotating disc dissolution rate methodology, which allows simultaneous determination of the dissolution rate of both active ingredient and polymer. The dissolution behavior of two bicalutamide:copovidone VA64 dispersion formulations, containing 5% (w/w) and 50% (w/w) bicalutamide, respectively, was investigated, with the aim of exploring the effect of increasing the bicalutamide loading on the dissolution performance. Spatially time-resolved Raman maps generated using multivariate curve resolution indicated the simultaneous transformation of amorphous bicalutamide present in the 50% drug-loaded extrudate into metastable polymorphic form II and low-energy polymorphic form I. Fitting a kinetic model and spatially correlating the data extracted from the Raman maps also allowed us to understand the re-crystallization mechanisms by which the low-energy form I appears. Form I was shown to crystallize mainly directly from the amorphous solid dispersion, with crystallization from the metastable form II being a minor contribution.

Determining the suitability of mass spectrometry for understanding the dissolution processes involved with pharmaceutical tablets.

RATIONALE: A current challenge for analytical chemists is the development of the measurement systems and approaches required to understand dynamic processes such as tablet dissolution. The design and development of oral tablets could be improved by the availability of detailed information about the rates of release of the individual tablet components. Small footprint mass spectrometry (MS) systems are gaining use for on-line reaction monitoring because of their ability to rapidly determine multiple reactant, intermediate, and product species. We have therefore assessed the utility of such MS systems to the study of dissolution processes. METHODS: Aqueous dissolution media containing phosphate and other non-volatile buffer salts were pumped from a standard USPII dissolution vessel through an active splitter and back. The splitter sampled the dissolution stream and diluted it into a make-up flow which was pumped to a small single quadrupole mass spectrometer. Single ion monitoring was used to quantify the ions of interest. Three different bio-relevant dissolution media were studied to gauge the effect of the sample matrix. RESULTS: Individual dissolution profiles were obtained from a tablet containing three drugs, and lactose as the soluble filler. This was successfully demonstrated with three different bio-relevant media designed to reflect the pH of the different sections of the human gastro-intestinal tract. Component concentrations as low as 0.06 µg/mL (representing 1% dissolution) were detected. The MS dissolution profiles correlated with the visual observation of tablet dissolution. MS gave linear responses with concentration for the individual components, although analysis of the tablet solution indicated that ion suppression is an area for further investigation. CONCLUSIONS: An on-line MS system was used to determine the individual dissolution profiles of three drugs and lactose as they were released from the same tablet. The level of each of these components in solution was determined every 10 seconds, and each had a similar release profile. The dissolution profiles were determined using inorganic buffer solutions at three different bio-relevant pHs.

Investigating the Dissolution Performance of Amorphous Solid Dispersions Using Magnetic Resonance Imaging and Proton NMR.

We have investigated the dissolution performance of amorphous solid dispersions of poorly water-soluble bicalutamide in a Kollidon VA64 polymeric matrix as a function of the drug loading (5% vs. 30% bicalutamide). A combined suite of state-of-the-art analytical techniques were employed to obtain a clear picture of the drug release, including an integrated magnetic resonance imaging UV-Vis flow cell system and 1H-NMR. Off-line 1H-NMR was used for the first time to simultaneously measure the dissolution profiles and rates of both the drug and the polymer from a solid dispersion. MRI and 1H-NMR data showed that the 5% drug loading compact erodes linearly, and that bicalutamide and Kollidon VA64 are released at approximately the same rate from the molecular dispersion. For the 30% extrudate, data indicated a slower water ingress into the compact which corresponds to a slower dissolution rate of both bicalutamide and Kollidon VA64.

Proton NMR: a new tool for understanding dissolution.

We present the use of (1)H NMR as a new measurement approach for improving understanding of the dissolution of pharmaceutical tablets. NMR has benefits over the conventional UV measurement approach in respect to much greater analyte selectivity and the ability to detect non-UV-absorbing species such as sugars. We used an in-line flow cell and water suppression experiments to determine the release profiles of three drug substances and lactose from the same tablet. Dissolution was performed in a pharmacopieal dissolution system with a standard protic buffer. NMR was shown to give high selectivity with each analyte having a well-resolved signal and sufficient sensitivity to determine the full release profile of even a compound present at only 5 mg in the tablet. The in-line flow cell gives excellent quality NMR spectra having little impact on peak shape. Dissolution of all the drug substances and lactose were determined to proceed at the same relative rates.

Development of Highly Sensitive Fluorescent Sensors for Separation-Free Detection and Quantitation Systems of Pepsin Enzyme Applying a Structure-Guided Approach.

Two fluorescent molecularly imprinted polymers (MIPs) were developed for pepsin enzyme utilising fluorescein and rhodamine b. The main difference between both dyes is the presence of two (diethylamino) groups in the structure of rhodamine b. Consequently, we wanted to investigate the effect of these functional groups on the selectivity and sensitivity of the resulting MIPs. Therefore, two silica-based MIPs for pepsin enzyme were developed using 3-aminopropyltriethoxysilane as a functional monomer and tetraethyl orthosilicate as a crosslinker to achieve a one-pot synthesis. Results of our study revealed that rhodamine b dyed MIPs (RMIPs) showed stronger binding, indicated by a higher binding capacity value of 256 mg g-1 compared to 217 mg g-1 for fluorescein dyed MIPs (FMIPs). Moreover, RMIPs showed superior sensitivity in the detection and quantitation of pepsin with a linear range from 0.28 to 42.85 µmol L-1 and a limit of detection (LOD) as low as 0.11 µmol L-1. In contrast, FMIPs covered a narrower range from 0.71 to 35.71 µmol L-1, and the LOD value reached 0.34 µmol L-1, which is three times less sensitive than RMIPs. Finally, the developed FMIPs and RMIPs were applied to a separation-free quantification system for pepsin in saliva samples without interference from any cross-reactors.

Developing a robust in vitro release method for a polymeric nanoparticle: Challenges and learnings.

Nanomedicines have emerged as a promising approach for targeted therapeutic delivery and specifically as a beneficial alternative to conventional cancer therapies as they can deliver higher concentrations of chemotherapeutic agents at the tumour site compared to healthy tissue, thus providing improved drug efficacy and lower systemic toxicity. Long acting injectables are increasingly becoming the focus of pharmaceutical research, as they can reduce dosing frequency and improve the life quality of patients. Development of an in vitro release (IVR) method for modified release nanomedicines is challenging because of the uniqueness and range of different formulation design approaches, as well as the complex nature of drug release mechanisms which may result in inherent variability. Regulatory guidance on the development of dissolution or release methods for parenteral products is limited relative to oral products. This article details the extensive in vitro release method development work conducted on a polymeric nanoparticle to develop the release media composition and selection of suitable apparatus and sampling technique to separate the released drug from the formulation. The aim was to develop a suitably robust analytical method that generated clinically relevant in vitro release data.

The Global Characterisation of a Drug-Dendrimer Conjugate - PEGylated poly-lysine Dendrimer.

The recent emergence of drug-dendrimer conjugates within pharmaceutical industry research and development introduces a range of challenges for analytical and measurement science. These molecules are very high molecular weight (100-200kDa) with a significant degree of structural complexity. The characteristics and quality attributes that require understanding and definition, and impact efficacy and safety, are diverse. They relate to the intact conjugate, the various building blocks of these complex systems and the level of the free and bound active pharmaceutical ingredient (API). From an analytical and measurement science perspective, this necessitates the measurement of the molecular weight, impurity characterisation, the quantitation of the number of conjugated versus free API molecules, the determination of the impurity profiles of the building blocks, primary structure and both particle size and morphology. Here we report the first example of a global characterisation of a drug-dendrimer conjugate - PEGylated poly-lysine dendrimer currently under development (AZD0466). The impact of the wide variety of analytical and measurement techniques on the overall understanding of this complex molecular entity is discussed, with the relative capabilities of the various approaches compared. The results of this study are an essential platform for the research and development of the future generations of related dendrimer-based medicines.

Linking dissolution to disintegration in immediate release tablets using image analysis and a population balance modelling approach.

PURPOSE: In order to achieve an improved understanding of disintegration and dissolution phenomena for an immediate release tablet formulation, a technique to monitor the number and size of particles entrained within the dissolution media was developed in combination with a population balancing model. METHODS: Tablets were first characterized for crushing force, disintegration time and dissolution performance using standard USP methodologies. The performance of the tablets was then assessed using a new measurement system which links a "QicPic" particle imaging device to a USP dissolution vessel. This system enables us to measure the number and size of particles generated during tablet dissolution. The population balance mathematical model allowed a tablet erosion rate to be manipulated to fit the experimental data. RESULTS: Results showed that tablets with differing crushing forces showed different dissolution behaviors that could be explained by differing rates of particle release into the dissolution media. These behaviors were then successfully modeled to provide a description of the dissolution and disintegration behavior of the tablets in terms of a tablet erosion rate. CONCLUSIONS: A new approach was developed that allowed the description of the dissolution behaviors of the tablets in terms of the rate that they release particles into solution. This was then successfully modeled in terms of a tablet erosion rate.

A computational chemistry-driven hypothesis on the mode of action of hipposudoric acid and related analogs.

Aim: To elucidate the mode of action of the hipposudoric acid derivatives and identify hit compounds for synthesis. Materials & methods: Structural fragments of known bioactive fluorenes were introduced onto the hipposudoric acid scaffold to yield novel derivatives. The binding motifs of the novel compounds were compared to the pharmacophore of DHFR co-crystallized with methotrexate (MTX). Results: Several of the novel compounds showed binding affinities that exceeded the affinity of the docked endogenous ligand (dihydrofolic acid). Conclusion: This study indicates that compounds 3r12, 3r9, 1s9 and 3r10 are promising candidates for synthesis and pharmacological evaluation.

Engineered Cas9 extracellular vesicles as a novel gene editing tool.

Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.

Discovery and SAR of 2-arylbenzotriazoles and 2-arylindazoles as potential treatments for Duchenne muscular dystrophy.

Families of 2-arylbenzotriazoles and 2-arylindazoles that show positive effects in screens predictive of endogenous utrophin upregulation have been identified. Synthesis and structure-activity relationships are described leading to compounds with attractive in vitro profiles.

Thiazolidinediones: An In-Depth Study of Their Synthesis and Application to Medicinal Chemistry in the Treatment of Diabetes Mellitus.

2,4-Thiazolidinedione (TZD) is a privileged and highly utilised scaffold for the development of pharmaceutically active compounds. This sulfur-containing heterocycle is a versatile pharmacophore that confers a diverse range of pharmacological activities. TZD has been shown to exhibit biological action towards a vast range of targets interesting to medicinal chemists. In this review, we attempt to provide insight into both the historical conventional and the use of novel methodologies to synthesise the TZD core framework. Further to this, synthetic procedures utilised to substitute the TZD molecule at the activated methylene C5 and N3 position are reviewed. Finally, research into developing clinical agents, which act as modulators of peroxisome proliferator-activated receptors gamma (PPARγ), protein tyrosine phosphatase 1B (PTP1B) and aldose reductase 2 (ALR2), are discussed. These are the three most targeted receptors for the treatment of diabetes mellitus (DM).