Long-acting nanoparticulate DNase-1 for effective suppression of SARS-CoV-2-mediated neutrophil activities and cytokine storm.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new strain of coronavirus not previously identified in humans. Globally, the number of confirmed cases and mortality rates of coronavirus disease 2019 (COVID-19) have risen dramatically. Currently, there are no FDA-approved antiviral drugs and there is an urgency to develop treatment strategies that can effectively suppress SARS-CoV-2-mediated cytokine storms, acute respiratory distress syndrome (ARDS), and sepsis. As symptoms progress in patients with SARS-CoV-2 sepsis, elevated amounts of cell-free DNA (cfDNA) are produced, which in turn induce multiple organ failure in these patients. Furthermore, plasma levels of DNase-1 are markedly reduced in SARS-CoV-2 sepsis patients. In this study, we generated recombinant DNase-1-coated polydopamine-poly(ethylene glycol) nanoparticulates (named long-acting DNase-1), and hypothesized that exogenous administration of long-acting DNase-1 may suppress SARS-CoV-2-mediated neutrophil activities and the cytokine storm. Our findings suggest that exogenously administered long-acting nanoparticulate DNase-1 can effectively reduce cfDNA levels and neutrophil activities and may be used as a potential therapeutic intervention for life-threatening SARS-CoV-2-mediated illnesses.

Evaluation of metabolism-mediated herb-drug interactions.

As the use of herbal medicines increases, the public health consequences of drug-herb interactions are becoming more significant. Herbal medicines share the same drug metabolizing enzymes and drug transporters, including cytochrome P450 enzymes (CYPs), glucuronosyltransferases (UGTs), and P-glycoprotein, with several clinically important drugs. Interactions of several commonly used herbal medicines, such as Ginko biloba, milk thistle, and St. John's wort, with therapeutic drugs including warfarin, midazolam, alprazolam, indinavir, saquinavir, digoxin, nifedipine, cyclosporine, tacrolimus, irinotecan, and imatinib in humans have been reported. Many of these drugs have very narrow therapeutic indices. As the herb-drug interactions can significantly alter pharmacokinetic and pharmacodynamic properties of administered drugs, the drugs interacting with herbal medicines should be identified by appropriate in vitro and in vivo methods. A good understanding of the mechanisms of herb-drug interactions is also essential for assessing and minimizing clinical risks. In vitro methods are useful for providing mechanistic information and evaluating multiple components in herbal medicines. This review describes major factors affecting the metabolism of herbal medicines, mechanisms of herb-drug interactions mediated by CYPs and UGTs, and several in vitro methods to assess the herb-drug interactions. Finally, drug interactions of Ginkgo biloba and St. John's wort, as representative herbal medicines, are described.

Quantitative determination of major phlorotannins in Ecklonia stolonifera.

Ecklonia stolonifera is a rich source of phlorotannins, which are responsible for the potent pharmacological effects associated with this seaweed. The purpose of this study was to develop a reversed-phase high-performance liquid chromatography method for the simultaneous determination of three major phlorotannins, eckol, dieckol, and phlorofucofuroeckol-A, in the extracts of Ecklonia stolonifera. The optimal chromatographic conditions were achieved on a Thermo Hypersil Gold C-18 column (250 x 4.6 mm i.d., 5 microm) using linear gradient elution of acetonitrile and water containing 0.1% formic acid at UV 254 nm. The separated phlorotannins were identified by liquid chromatography-mass spectrometry. The high-performance liquid chromatography method showed good linearity (r2 > 0.998), precision (1.4-9.5%), and accuracy (93.9-108.7%). The limits of detection ranged from 0.06 to 0.30 microg/mL and the lower limits of quantitation ranged from 0.2 to 1.0 microg/mL. Among phlorotannins, dieckol was the most abundant in both ethanol and ethyl acetate extracts of Ecklonia stolonifera.

Simultaneous determination of quercetin and its glycosides from the leaves of Nelumbo nucifera by reversed-phase high-performance liquid chromatography.

The purpose of this study was to develop reversed-phase HPLC method for the simultaneous determination of the flavonoids from the leaves of Nelumbo nucifera, which have been known to exhibit antioxidant, anti-HIV, antihyperlipidemic and antiobesity effects. HPLC separation was achieved on C(18) column using gradient elution with mobile phase consisting of acetonitrile-water containing 0.1% formic acid. The separated peaks were identified by electrospray ionization mass spectrometry. The HPLC method was validated and applied for the simultaneous determination of the bioactive flavonoids from the leaves of Nelumbo nucifera. In the methanol extract, six flavonoids, quercetin, rutin, quercetin 3-O-beta-D-galactopyranoside (Qc-3-Gal), quercetin 3-O-beta-D-glucopyranoside (Qc-3-Glc), quercetin 3-O-beta-D-glucuronide (Qc-3-Gln) and quercetin 3-O-alpha-arabinopyranosyl-(1-->2)-beta-galactopyranoside (Qc-3-AraGal), were identified. Among them, Qc-3-Glc and Qc-3-Gln were found to be major component in the methanol extract of Nelumbo nucifera leaves.

PEGylation of octreotide: II. Effect of N-terminal mono-PEGylation on biological activity and pharmacokinetics.

PURPOSE: : To determine the optimal polyethylene glycol (PEG)-conjugate of octreotide by evaluating the effects of PEGylation chemistry on the biological activity and pharmacokinetic properties. METHODS: : Octreotide was chemically modified by reaction with succinimidyl propionate monomethoxy PEG (SPA-mPEG, molecular weight 2000) or succinimidyl butyraldehyde-mPEG (ALD-mPEG, molecular weight 2000 and 5000). The structural conformation of PEG-octreotides was evaluated by circular dichroism (CD), the biological activity was assessed by measuring the decrease of serum insulin-like growth factor-I levels in rats, and a pharmacokinetic study was performed after subcutaneous administration in rats. The stability against acylation was investigated with poly(D,L -lactide-co-glycolide) (PLGA). RESULTS: : ALD-mPEG was site-specific in PEGylating octreotide at the N-terminus. The mono-PEG-octreotides prepared with ALD-mPEG (mono-ALDPEG-octreotide), which alkyl bond preserves the amine's positive charge, showed complete preservation of biological activity, whereas the PEG-octreotides prepared with SPA-mPEG showed lower activity. In the CD analysis, the spectra of the mono-ALDPEG-octreotides were nearly superimposable with that of native octreotide. The mono-ALDPEG-5K-octreotide showed significantly improved pharmacokinetic properties compared with mono-ALDPEG-2K-octreotide as well as native octreotide. Both mono-ALDPEG-2K- and mono-ALDPEG-5K-octreotides were stable against acylation by degrading PLGA. CONCLUSIONS: : The mono-PEGylation of octreotide at N-terminus with ALD-mPEG produced a conjugate that is biologically and structurally active and stable against acylation by PLGA, and therefore it may serve as a candidate for somatostatin microsphere formulations.

Monitoring of peptide acylation inside degrading PLGA microspheres by capillary electrophoresis and MALDI-TOF mass spectrometry.

The purpose of this research was to assess the acylation reactions of peptides, salmon calcitonin (sCT), human parathyroid hormone 1-34 (hPTH1-34) and leuprolide, in poly(lactic-co-glycolic acid) (PLGA) microspheres. Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used for determining and monitoring peptide acylation and quantitating acylation products in the degrading PLGA microspheres. In the degrading PLGA microspheres of sCT and hPTH1-34, the acylation products were observed and determined to be adducts with glycolic acid units from degradable PLGA polymer by MALDI-TOF MS. In the microsphere of leuprolide, however, the acylation product was not observed even after 28 days of incubation at the release medium, which represents the different stabilities among peptides according to the primary structure. As the leuprolide contains tyrosine and serine having hydroxyl group of nucleophilic amino acids, the acylation reaction of peptide is shown to be mainly due to the primary amino groups of N-terminus or lysine residue. The complementary use of CE and MALDI-TOF MS will be useful for searching the counter measures as well as determining the peptide acylation in the manufactured formulations on the market.