A New Practice to Monitor the Fabrication Process of Fab-Targeting Ligands from Bevacizumab by LC-MS: Preparation and Analytical Characterization

The objective of this study was to qualitatively evaluate a Fab-targeting ligand preparation containing free thiol groups in the hinge region by using bevacizumab as a model. The evaluation focused on the purification of fragments through a nonaffinity-based process using a centrifugal ultrafiltration technique and mild reduction conditions for the intact production of F(ab’) fragments with specific inter-heavy-chain disulfide bonds cleavage. Under these conditions, F(ab’) fragments with a defined chemical composition were successfully obtained via proteolytic digestion followed by a controlled reduction reaction process maintaining the integrity of the binding sites. The ultrafiltration purification technique appears to be suitable for the removal of the digestive enzyme but inefficient for the removal of Fc fragments, thus requiring additional processing. A suitable analytical strategy was developed, allowing us to demonstrate the reformation of disulfide bridges between the two reduced cysteines within F(ab’) fragments.

Traditional Uses, Phytochemistry, and Pharmacology of Elegia Species: A Review

In South Africa, plants belonging to the Restionaceae family possess an ecological dominance. As a result, they have been the subject of numerous morphological, anatomical, and evolutionary studies. However, few studies have focused on their phytochemical profile and their potential pharmacological activities. The genus Elegia L. is the second largest of this family comprising 52 species, which are mainly used as materials for thatching. Limited studies on the chemical constituents of Elegia species and their importance as medicinal plants have been undertaken. This review provides constructive and extensive information about the botanical characterization, distribution, traditional uses, phytochemistry and pharmacology of the genus Elegia. A comprehensive search of previously published literature was performed for studies on this genus, using databases with different key search words. This survey documented 52 Elegia species summarizing their previous taxonomic classification. In addition, 14 species were found to be studied for their phytochemical profile, revealing 14 chemical compounds. Concerning their biological activities, only one species (E. tectorum (L.f.) Moline and H.P.Linder) is reported for its anti-wrinkle activity. Moreover, two species are locally used for thatching and as materials for brooms. The present review highlights the Elegia genus as an important source of bioactive phytochemicals with flavonol glycosides being the main metabolites and reveals the uncharted territory of this genus for new research studies.

The Development and the Validation of a Novel Dissolution Method of Favipiravir Film-Coated Tablets

The aim of this study was to develop and validate a dissolution test for favipiravir release in a tablet dosage form using ultra-high performance liquid chromatography (UHPLC). The dissolution method was developed by testing the solubility of favipiravir in media with different pH values. The results demonstrated that the best dissolution was achieved in phosphate buffer with a pH of 6.8. The amount of favipiravir that was released was about 100% after 30 min. The UHPLC method presented linearity (R = 1.000) in the concentration range of 0.044–0.44 mg/mL. The recovery parameter that was achieved ranged from 102.5% to 104.2%. The system suitability, repeatability, and intermediate precision RSD% results were found to be 0.36%, 1.99%, and 2.49%, respectively. In addition to these parameters and results, an F-test was performed using the Minitab 18 Statistical Software program for the intermediate precision and repeatability results. The standard and sample solutions were found to be stable for 2 days in their respective dissolution medium. This analytical method was also found to be selective for favipiravir. In conclusion, a simple and feasible dissolution method with a short run time of 2.5 min was developed and validated successfully. The obtained results demonstrated that the dissolution test developed here is adequate for its purpose and can be applied as the dissolution method for favipiravir in film-coated tablets for release analyses.

The Search for New Antibacterial Agents among 1,2,3-Triazole Functionalized Ciprofloxacin and Norfloxacin Hybrids: Synthesis, Docking Studies, and Biological Activity Evaluation

Among all modern antibiotics, fluoroquinolones are well known for their broad spectrums of activity and efficiency toward microorganisms and viruses. However, antibiotic resistance is still a problem, which has encouraged medicinal chemists to modify the initial structures in order to combat resistant strains. Our current work is aimed at synthesizing novel hybrid derivatives of ciprofloxacin and norfloxacin and applying docking studies and biological activity evaluations in order to find active promising molecules. We succeeded in the development of a synthetic method towards 1,2,3-triazole-substituted ciprofloxacin and norfloxacin derivatives. The structure and purity of the obtained compounds were confirmed by 1H NMR, 13C NMR, 19F NMR, LC/MS, UV-, IR- spectroscopy. Docking studies, together with in vitro research against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853, Candida albicans NCTC 885-653 revealed compounds in which activity exceeded the initial molecules.

Using Metabolite Data to Develop Patient Centric Specification for Amide Impurity in Vildagliptin Tablets

Many specified impurities in vildagliptin's finished product have been disclosed in the literature that are above their qualification threshold. We used the impurity B (amide impurity) as a case example to explore whether existing literature can be leveraged to determine the safe level of impurity and thereby develop a patient-centric specification (PCS) for impurities. No-observed-adverse-effect level (NOAEL) was derived from rate metabolism information and converted to human equivalent dose (HED). The HED was estimated as 6.5 mg/day. The high qualification levels are supported by repeat dose toxicity studies performed in rats, mice and dogs. Maximum theoretical amount (MTA) was correlated with the maximum observed amount (MOA) to verify whether the exposure was due to impurity and/or metabolite. MOA/MTA was found ≥1 suggesting that metabolism contributed to the amount excreted in feces and therefore could be used to further justify a higher specification limit than the usual one of ≤0.5%. Quite often the drug metabolism and degradation pathways overlap, resulting in the formation of identical constituents. Therefore, metabolism data can be leveraged for deriving safe levels of degradation impurities and develop PCS for impurities.

Synthesis and Biological Activity Evaluation of Novel 5-Methyl-7-phenyl-3H-thiazolo[4,5-b]pyridin-2-ones

A series of 5-methyl-7-phenyl-3H-thiazolo[4,5-b]pyridin-2-ones has been designed, synthesized, and characterized by spectral data. Target compounds were screened for their antimicrobial activity against some pathogenic bacteria and fungi, and most of them showed moderate activity, especially compound 3g, which displayed the potent inhibitory effect against Pseudomonas aeruginosa and Escherichia coli with MIC value of 0.21 μM. The active thiazolopyridine derivatives 3c, 3f, and 3g were screened for their cytotoxicity effects on HaCat, Balb/c 3T3 cells using MTT assay, which revealed promising results. In silico assessment for compounds 3c, 3f, and 3g also revealed suitable drug-like parameters and ADME properties. The binding interactions of the most active compound 3g were performed through molecular docking against MurD and DNA gyrase, with binding energies and an inhibitory constant compared to the reference drug ciprofloxacin. The tested thiazolo[4,5-b]pyridines constitute an exciting background for the further development of new synthetic antimicrobial agents.

Compatibility of Different Formulations in Pentravan® and Pentravan® Plus for Transdermal Drug Delivery

The potential therapeutic benefit of transdermal delivery systems for some active pharmaceutical ingredients (APIs) has been well-established for decades within the scientific community. However, together with the clinical efficacy, there is the need for an evaluation of the stability of such APIs in bases with known transdermal capabilities, which is necessary to provide the compounding pharmacist with confidence when providing transdermal products. In this study, the stability of danazol, metformin HCl, and resveratrol as individual ingredients, as well as metformin HCl, resveratrol, and Vitamin D3 in combinations at bracketed high and low concentrations, were evaluated over a period of 6 months, using a ready-to-use transdermal vehicle for compounding pharmacies (Pentravan® or Pentravan® Plus). The five formulations tested (F1: Danazol 50 mg/g + MiodesinTM 85 mg/g in Pentravan®, F2: Metformin HCl 200 mg/g in Pentravan®, F3: Resveratrol 200 mg/g in Pentravan®, F4: Metformin HCl 100 mg/g + Resveratrol 100 mg/g + Vitamin D3 5000 IU in Pentravan®, and F5: Metformin HCl 200 mg/g + Resveratrol 200 mg/g + Vitamin D3 5000 IU in Pentravan® Plus) presented a beyond-use date of at least 6 months, presenting high convenience for the compounding pharmacies.

Phytochemistry and Evidence-Based Traditional Uses of the Genus Achillea L.: An Update (2011–2021)

Knowledge within the field of phytochemistry research has accelerated at a tremendous speed. The excess of literature reports featuring plants of high ethnopharmacological importance, in combination with our interest in the Asteraceae family and traditional medicine, led us to acknowledge the value of the Achillea L. genus. In a broad context, the various Achillea species are used around the globe for the prevention and treatment of different diseases, including gastrointestinal problems, haemorrhages, pneumonia, rheumatic pains, diuresis, inflammation, infections, and wounds, as well as menstrual and gynaecologic abnormalities. The present review aims to provide and summarize the recent literature (2011–2021) on the phytochemistry of the Achillea genus. In parallel, this study attempts to bridge the reports on the traditional uses with modern pharmacological data. Research articles that focused on secondary metabolites, traditional uses and pharmacological activities were collected from various scientific databases such as Pubmed, ScienceDirect, Reaxys and Google Scholar. This study revealed the presence of 141 phytochemicals, while 24 traditionally used Achillea spp. were discussed in comparison to current data with an experimental basis.

Design, Synthesis and In Vitro Antimicrobial Activity of 6-(1H-Benzimidazol-2-yl)-3,5-dimethyl-4-oxo-2-thio-3,4-dihydrothieno[2,3-d]pyrimidines

The rapid development in bacterial resistance to many groups of known antibiotics forces the researchers to discover antibacterial drug candidates with previously unknown mechanisms of action, one of the most relevant being the inhibition of tRNA (Guanine37-N1)-methyltransferase (TrmD). The discovery of selective TrmD inhibitors in the series of carboxamide derivatives of thienopyrimidines became a background for further modification of the similar structures aimed at the development of promising antibacterial agents. As part of this research, we carried out the construction of heterocyclic hybrids bearing the moieties of thieno[2,3-d]pyrimidine and benzimidazole starting from 3,5-dimethyl-4-oxo-2-thioxo-1H-thieno[2,3-d]pyrimidine-6-carboxylic acid, which was used as the pivotal intermediate. The hybrid molecule of 6-(1H-benzimidazol-2-yl)-3,5-dimethyl-2-thioxo-1H-thieno[2,3-d]pyrimidin-4-one prepared via condensation of the carboxylic acid with ortho-phenylenediamine was further alkylated with aryl/hetaryl chloroacetamides and benzyl chloride to produce the series of S-alkyl derivatives. The results of molecular docking studies for the obtained series of S-alkyl benzimidazole-thienopyrimidines showed their high affinity to the TrmD isolated from the P. aeruginosa. The results of antimicrobial activity screening revealed the antimicrobial properties for all of the studied molecules against both Gram-positive and Gram-negative bacteria and the Candida albicans fungal strain. The highest antimicrobial activity was determined for 2-{[6-(1H-benzimidazol-2-yl)-3,5-dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl]thio}-N-(4-isopropylphenyl)acetamide, which also had the highest affinity to the TrmD inhibitor’s binding site according to the docking studies results.

Purification and Biochemical Characterization of Taxadiene Synthase from Bacillus koreensis and Stenotrophomonas maltophilia

Taxadiene synthase (TDS) is the rate-limiting enzyme of Taxol biosynthesis that cyclizes the geranylgeranyl pyrophosphate into taxadiene. Attenuating Taxol productivity by fungi is the main challenge impeding its industrial application; it is possible that silencing the expression of TDS is the most noticeable genomic feature associated with Taxol-biosynthetic abolishing in fungi. As such, the characterization of TDS with unique biochemical properties and autonomous expression that is independent of transcriptional factors from the host is the main challenge. Thus, the objective of this study was to kinetically characterize TDS from endophytic bacteria isolated from different plants harboring Taxol-producing endophytic fungi. Among the recovered 23 isolates, Bacillus koreensis and Stenotrophomonas maltophilia achieved the highest TDS activity. Upon using the Plackett–Burman design, the TDS productivity achieved by B. koreensis (18.1 µmol/mg/min) and S. maltophilia (14.6 µmol/mg/min) increased by ~2.2-fold over the control. The enzyme was purified by gel-filtration and ion-exchange chromatography with ~15 overall folds and with molecular subunit structure 65 and 80 kDa from B. koreensis and S. maltophilia, respectively. The chemical identity of taxadiene was authenticated from the GC-MS analyses, which provided the same mass fragmentation pattern of authentic taxadiene. The tds gene was screened by PCR with nested primers of the conservative active site domains, and the amplicons were sequenced, displaying a higher similarity with tds from T. baccata and T. brevifolia. The highest TDS activity by both bacterial isolates was recorded at 37–40 °C. The Apo-TDSs retained ~50% of its initial holoenzyme activities, ensuring their metalloproteinic identity. The activity of purified TDS was completely restored upon the addition of Mg2+, confirming the identity of Mg2+ as a cofactor. The TDS activity was dramatically reduced upon the addition of DTNB and MBTH, ensuring the implementation of cysteine-reactive thiols and ammonia groups on their active site domains. This is the first report exploring the autonomous robust expression TDS from B. koreensis and S. maltophilia with a higher affinity to cyclize GGPP into taxadiene, which could be a novel platform for taxadiene production as intermediary metabolites of Taxol biosynthesis.