Phytochemical screening of Cordia glabrata (MART.) A.DC. extracts and its potential antioxidant, photoprotective, antimicrobial and antiviral activities

Species of the genus Cordia have shown biological activities, such as anti-inflammatory, analgesic, antioxidant, antiviral, and antifungal activities. The species Cordia glabrata (MART) A.DC. Has no information concerning its phytochemical profile and possible biological activities. Thus, this study aimed to evaluate this profile in ethanolic extracts of young, adult and senescent leaves, as well as their antioxidant, photoprotective, antimicrobial, and virucidal potentials. Phytochemical analysis was performed by TLC (thin-layer chromatography) and showed the presence of flavonoids, tannins, and terpenes. The evaluation by UPLC-MS/MS (Ultra performance liquid chromatography - tandem mass spectrometer) evidenced the presence of caffeic (3.89 mgL-1), p-cumaric (6.13 mgL-1), and ferulic (0.58 mgL-1) acids, whilst, in GC/MS (Gas chromatography–mass spectrometry) analysis there was a greater amount of palmitic (51.17%), stearic (20.34%), linoleic (9.62%), and miristic (8.16%) fatty acids. The DPPH (2,2-Diphenyl-1-picrylhydrazyl) and ABTS+ (2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radicals were used to verify the potential antioxidant activity, observing a better activity for the leaf extract in the adult phenological stage: 54.63 ± 1.06 µgmL-1 (DPPH) and 44.21 ± 1.69 mM (ABTS). The potential photoprotective activity of the extracts was determined by spectrophotometry and the in vitro values of SPF (Sun Protection Factor) in young and adult leaves (5.47 and 5.41, respectively) showed values close to the minimum SPF of 6.0 required by ANVISA (Brazilian Health Regulatory Agency). It was not observed an antimicrobial activity for Staphylococcus aureus with a minimum inhibitory concentration of 2000 μgmL-1, however the anti-herpetic assay against the Herpes simplex virus type 2 (HSV-2) showed a potent virucidal activity at the tested concentrations with CV50 value <0.195 μgmL-1 and a Selectivity Index (SI = CC50 / CV50) greater than 448. The results obtained in this study suggest that extracts of leaves of C. glabrata in their adult phenological stage have potential antioxidant, photoprotective and virucidal activity, considering in vitro test results.

Single-cell analysis reveals selection of TP53-mutated clones after MDM2 inhibition

The mechanisms of transformation of chronic myeloproliferative neoplasms (MPN) to leukemia are largely unknown but TP53mutations acquisition is considered a key event in this process. P53 is a main tumor suppressor but mutations in this protein per se do not confer a proliferative advantage to the cells and a selection process is needed for the expansion of mutant clones. MDM2 inhibitors may rescue normal p53 from degradation and have been evaluated in a variety of cancers with promising results. However the impact of these drugs on TP53 mutated cells is underexplored. We report herein evidence of a direct effect of MDM2 inhibition on the selection of MPN patients' cells harboring TP53 mutations. To decipher whether these mutations can arise in a specific molecular context we used a DNA single cell approach to determine the clonal architecture of TP53 mutated cells. We observed that TP53 mutations are late events in MPN mainly occurring in the driver clone while clonal evolution frequently consists of sequential branching instead of linear consecutive acquisition of mutations in the same clone. At the single cell level the presence of additional mutations does not influence the selection of TP53 mutant cells by MDM2 inhibitor treatment. Also, we describe an in vitro test allowing to predict the emergence of TP53 mutated clones. Altogether, this is the first demonstration that a drug treatment can directly favor the emergence of TP53-mutated subclones in MPN.

Impact of gastrointestinal differences in veterinary species on the oral drug solubility, in vivo dissolution, and formulation of veterinary therapeutics

Many gaps exist in our understanding of species differences in gastrointestinal (GI) fluid composition and the associated impact of food intake and dietary composition on in vivo drug solubilization. This information gap can lead to uncertainties with regard to how best to formulate pharmaceuticals for veterinary use or the in vitro test conditions that will be most predictive of species-specific in vivo oral product performance. To address these challenges, this overview explores species-specific factors that can influence oral drug solubility and the formulation approaches that can be employed to overcome solubility-associated bioavailability difficulties. These discussions are framed around some of the basic principles associated with drug solubilization, reported species differences in GI fluid composition, types of oral dosage forms typically given for the various animal species, and the effect of prandial state in dogs and cats. This basic information is integrated into a question-and-answer section that addresses some of the formulation issues that can arise in the development of veterinary medicinals.

Oligochitosan Synthesized by Cunninghamella elegans, a Fungus from Caatinga (The Brazilian Savanna) Is a Better Antioxidant than Animal Chitosan

Animal chitosan (Chit-A) is gaining more acceptance in daily activities. It is used in a range of products from food supplements for weight loss to even raw materials for producing nanoparticles and hydrogel drug carriers; however, it has low antioxidant activity. Fungal oligochitosan (OChit-F) was identified as a potential substitute for Chit-A. Cunninghamella elegans is a fungus found in the Brazilian savanna (Caatinga) that produces OligoChit-F, which is a relatively poorly studied compound. In this study, 4 kDa OChit-F with a 76% deacetylation degree was extracted from C. elegans. OChit-F showed antioxidant activity similar to that of Chit-A in only one in vitro test (copper chelation) but exhibited higher activity than that of Chit-A in three other tests (reducing power, hydroxyl radical scavenging, and iron chelation). These results indicate that OChit-F is a better antioxidant than Chit-A. In addition, Chit-A significantly increased the formation of calcium oxalate crystals in vitro, particularly those of the monohydrate (COM) type; however, OChit-F had no effect on this process in vitro. In summary, OChit-F had higher antioxidant activity than Chit-A and did not induce the formation of CaOx crystals. Thus, OChit-F can be used as a Chit-A substitute in applications affected by oxidative stress.

In vitro tools for orally inhaled drug products—state of the art for their application in pharmaceutical research and industry and regulatory challenges

The drug development process is a lengthy and expensive challenge for all involved players. Experience with the COVID-19 pandemic underlines the need for a rapid and effective approval for treatment options. As essential prerequisites for successful drug approval, a combination of high-quality studies and reliable research must be included. To this day, mainly in vivo data are requested and collected for assessing safety and efficacy and are therefore decisive for the pre-clinical evaluation of the respective drug. This review aims to summarize the current state of the art for safety and efficacy studies in pharmaceutical research and industry to address the relevant regulatory challenges and to provide an outlook on implementing more in vitro methods as alternative to animal testing. While the public demand for alternative methods is becoming louder, first examples have meanwhile found acceptance in relevant guidelines, e.g. the OECD guidelines for skin sensitizer. Besides ethically driven developments, also the rather low throughput and relatively high costs of animal experiments are forcing the industry towards the implementation of alternative methods. In this context, the development of orally inhaled drug products is particularly challenging due to the complexity of the lung as biological barrier and route of administration. The replacement of animal experiments with focus on the lungs requires special designed tools to achieve predictive data. New in vitro test systems of increasing complexity are presented in this review. Limits and advantages are discussed to provide some perspective for a future in vitro testing strategy for orally inhaled drug products. Graphical abstract

The Research of Toxicity and Sensitization Potential of PEGylated Silver and Gold Nanomaterials

Polyethylene glycol (PEG) is a polymer used for surface modification of important substances in the modern pharmaceutical industry and biopharmaceutical fields. Despite the many benefits of PEGylation, there is also the possibility that the application and exposure of the substance may cause adverse effects in the body, such as an immune response. Therefore, we aimed to evaluate the sensitization responses that could be induced through the intercomparison of nanomaterials of the PEG-coated group with the original group. We selected gold/silver nanomaterials (NMs) for original group and PEGylated silver/gold NMs in this study. First, we measured the physicochemical properties of the four NMs, such as size and zeta potential under various conditions. Additionally, we performed the test of the NM’s sensitization potential using the KeratinoSens™ assay for in vitro test method and the LLNA: 5-bromo-2-deoxyuridine (BrdU)-FCM for in vivo test method. The results showed that PEGylated-NMs did not lead to skin sensitization according to OECD TG 442 (alternative test for skin sensitization). In addition, gold nanomaterial showed that cytotoxicity of PEGylated-AuNMs was lower than AuNMs. These results suggest the possibility that PEG coating does not induce an immune response in the skin tissue and can lower the cytotoxicity of nanomaterials.

In-Body Electromagnetic Sensor Combined with AI-Enhanced Electrocardiography for Pacemaker Working Status Telemonitoring

This paper describes the design and implementation of an in-body electromagnetic sensor for patients with implanted pacemakers. The sensor can either be mounted on myocardial tissue and monitor the electrocardiography (ECG) with contact electrodes or implanted under the skin and monitor the ECG with coaxial leads. A 16-bit high-resolution analog front-end (AFE) and an energy-efficient 32-bit CPU are used for instantaneous ECG recording. Wireless data transmission between the sensor and clinician’s computer is achieved by an embedded low-power Bluetooth transmitter. In order to automatically recognize the working status of the pacemaker and alarm the episodes of arrhythmias caused by pacemaker malfunctions, pacing mode classification and fault diagnosis on the recorded ECG were achieved based on an AI algorithm, i.e., a resource allocation network (RAN). A prototype of the sensor was implemented on a human torso, and the in vitro test results prove that the sensor can work properly for the 1-4-meter transmission range.

Comparative In vitro Metabolism of Enflicoxib in Dogs, Rats, and Humans: Main Metabolites and Proposed Metabolic Pathways

Background: Enflicoxib is a non-steroidal anti-inflammatory drug of the coxib family characterized by a long-lasting pharmacological activity that has been attributed to its active metabolite E-6132. Objective: The aim of this work was to explore enflicoxib biotransformation in vitro in humans, rats and dogs, and to determine its metabolic pathways. Method: Different in vitro test systems were used, including hepatocytes and liver and non-hepatic microsomes. The samples were incubated with enflicoxib and/or any of its metabolites at 37°C for different times depending on the test system. The analyses were performed by liquid chromatography coupled with either radioactivity detection or high-resolution mass spectrometry. Results: Enflicoxib was efficiently metabolized by cytochrome P-450 into three main phase I metabolites: M8, E-6132, and M7. The non-active hydroxy-pyrazoline metabolite M8 accounted for most of the fraction metabolized in all the three species. The active pyrazol metabolite E-6132 showed a slow formation rate, especially in dogs, whereas metabolite M7 was a secondary metabolite formed by oxidation of M8. In hepatocytes, diverse phase II metabolite conjugates were formed, including enflicoxib glucuronide, M8 glucuronide, E-6132 glucuronide, M7 glucuronide, and M7 sulfate. Metabolite E-6132 was most probably eliminated by a unique glucuronidation reaction at a very low rate. Conclusion: The phase I metabolism of enflicoxib was qualitatively very similar among rats, humans and dogs. The low formation and glucuronidation rates of the active enflicoxib metabolite E-6132 in dogs are postulated as key factors underlying the mechanism of its long-lasting pharmacokinetics and enflicoxib's overall sustained efficacy.

Tackling penicillin allergy delabeling with ultra-sensitive in vitro test and human-like specific IgE standards

Background: Penicillin allergy delabeling initiatives are now part of antibiotic stewardship programs and include the use of invasive and risky in vivo tests. Instead, the quantification of specific IgE is highly useful to confirm immediate allergy to penicillins. However, discrepant results associated to the low sensitivity of the in vitro tests have limited their routine diagnostic use for delabeling purposes. We aimed to tackle a novel diagnostic strategy for specific IgE testing based on a homologous interpolation scheme, using recombinantly produced standards. Methods: Serum samples from a cohort of allergic patients and controls were analysed by a chemiluminescence-based immunoassay, using a bispecific binanobody as standard. The novel standard targets the major antigenic determinant of penicillin G and the paratope of Omalizumab, acting as human-like specific IgE. Results: Testing a cohort of 65 human serum samples, the method achieved a good agreement and strong positive relationship, reaching a limit of detection below 0.1 IU/mL. The sensitivity of the in vitro test significantly increased (66 %), doubling that of the ImmunoCAP reference in vitro assay with an overall specificity of 100 %. Conclusions: The new diagnostic strategy compares favourably with the results obtained on the ImmunoCAP system, paving the way towards the standardization of penicillin allergy testing. The recombinant standards are potent calibrators, highly stable, easy and inexpensive to produce, and overcome the limitation associated with preparations derived from pooled human serum, expediting the production of next generation standards with different specificities to successfully tackle β-lactam allergy delabeling by in vitro tests.