Zhiguo Ma

Identification of glucuronidation and biliary excretion as the main mechanisms for gossypol clearance: in vivo and in vitro evidence

Abstract 1. The natural polyphenol gossypol possesses many therapeutic benefits. Here we aim to determine the elimination pathways of gossypol in vivo and in vitro. 2. Metabolite elucidation of gossypol was performed using UPLC-QTOF/MS coupled with Metabolynx analysis. Clearance of gossypol was evaluated in bile duct cannulated rats and in the single-pass perfused rat intestine model. In vitro glucuronidation of gossypol was characterized using liver and intestine microsomes as well as recombinant UDP-glucuronosyltransferase (UGT) enzymes. 3. Analysis of rat plasma, urine, and feces revealed glucuronidation as the only metabolic pathway for gossypol. In bile duct cannulated rats, considerable amounts of glucuronides (G1, G2 and G3; 58.8–83.2% of dose) and parent compound (5.0–20%) were excreted into bile after IV administration. In the perfused rat intestine model, gossypol was well absorbed with a (the dimensionless effective permeability) value of 4.4. Significant amounts of glucuronides (G1, G2 and G3) were excreted into the gut lumen (2.5%) and into the bile (4.8%). Biliary excretion of unchanged gossypol (6.0%) was comparable to that of glucuronides. Further, gossypol was subjected to rapid glucuronidation by liver and intestine microsomes. Reaction phenotyping showed that multiple UGT1A enzymes (including UGT1A1, 1A3, 1A7 and 1A8) are mainly responsible for gossypol metabolism. 4. In conclusion, glucuronidation was the only metabolic pathway for gossypol in rats. Excretion of unchanged gossypol into bile was also an important clearance mechanism.

Enhancement of Oral Bioavailability of Tripterine Through Lipid Nanospheres: Preparation, Characterization, and Absorption Evaluation

Oral delivery of anticancer drugs remains challenging because of limited water-solubility and/or poor permeability. Here, we aimed to enhance the oral bioavailability of tripterine (TRI, a plant-derived anticancer compound) using lipid nanospheres (LNs) and to determine the mechanisms of oral absorption. TRI-loaded LNs (TRI-LNs) were prepared by rapid dispersion of an ethanol mixture of TRI, lecithin, sodium oleate, and soybean oil into water. The obtained LNs were 150 nm in size with a high value of entrapment efficiency (99.95%). TRI-LNs were fairly stable and the drug release was negligible (<0.2%) in simulated physiological fluid. The pharmacokinetic results showed that LNs significantly enhanced the oral bioavailability of TRI with a relative bioavailability of 224.88% (TRI suspensions was used as a reference). The mechanistic studies demonstrated that improved intestinal permeability and post-enterocyte lymphatic transport were mainly responsible for the enhanced oral absorption. Our findings suggested that LNs may be a viable oral carrier for poorly bioavailable drugs.

Metabolite elucidation of the Hsp90 inhibitor SNX-2112 using ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS)

Abstract 1. The novel heat-shock protein 90 inhibitor SNX-2112 is a promising drug candidate for treating various types of cancers. Here we aim to determine the metabolic pathways of SNX-2112 in rats in vivo and in humans in vitro. 2. Metabolite identification was performed using ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) method. In vitro metabolism studies were performed using liver and intestine microsomes, as well as recombinant human cytochrome P450 (CYP) enzymes. 3. Analysis of rat plasma, urine, and feces revealed a total of eight metabolites, one reductive metabolite (M1), one structurally unknown metabolite (M2), and six mono-oxidative metabolites (M3-1, M3-2, M3-3, M3-4, M3-5, and M3-6). The reduction, M2, and mono-oxidation pathways were responsible for 0.8 ± 0.3 %, 18.3 ± 9.1 %, and 39.4% ± 6.1 of SNX-2112 clearance from rats, respectively. 4. SNX-2112 was subjected to the same types of metabolism in human liver and intestine microsomes. Reaction phenotyping showed that CYP3A4, 3A5, 2D6, and 1A1 were mainly responsible for SNX-2112 metabolism. 5. In conclusion, we have elucidated the metabolic pathways of SNX-2112 and highlighted that metabolism was the predominant pathway for its clearance. Better understanding of SNX-2112 metabolism should facilitate the drug development of this promising anti-cancer agent.

Effects of pharmaceutical PEGylation on drug metabolism and its clinical concerns

Introduction: PEGylation refers to covalent conjugation of one or more polyethylene glycol chains to a drug molecule. It also refers to formulation of a drug into PEGylated drug-delivery vehicles in pharmacy. It is well-known that PEGylation can greatly influence the pharmacokinetics and pharmacodynamics of drugs. Areas covered: This article describes the importance of PEGylation in drug development and research. The impact of PEGylation on drug metabolism and the clinical safety of PEGylated drugs (or formulations) are also discussed. Information and data from the literature were collected, analyzed and summarized. Expert opinion: PEGylation is an effective approach to potentiate drugs with undesirable properties. Currently, PEGylation has penetrated into every field of pharmaceutical practice, involving biomacromolecules, small drugs and drug delivery systems. Efficacy enhancement is attained through modification of the pharmacokinetics and toxicity profiles of parent drugs. As a result of PEGylation, the drugs tend to display enhanced solubility, prolonged circulatory time and reduced immunogenicity/antigenicity. The bottleneck of PEGylation is how to break through the limitation of chemical conjugation and to properly preserve the pharmacological activity of the drug. PEGylated formulation is an area deserving more attention in terms of systemic delivery of insoluble small drugs.

Stable Knock-down of Efflux Transporters Leads to Reduced Glucuronidation in UGT1A1-Overexpressing HeLa Cells: The Evidence for Glucuronidation-Transport Interplay

Efflux of glucuronide is facilitated by the membrane transporters including BCRP and MRPs. In this study, we aimed to determine the effects of transporter expression on glucuronide efflux and cellular glucuronidation. Single efflux transporter (i.e., BCRP, MRP1, MRP3, or MRP4) was stably knocked-down in UGT1A1-overexpressing HeLa cells. Knock-down of transporters was performed by stable transfection of short-hairpin RNA (shRNA) using lentiviral vectors. Glucuronidation and glucuronide transport in the cells were characterized using three different aglycones (i.e., genistein, apigenin, and emodin) with distinct metabolic activities. BCRP knock-down resulted in significant reductions in excretion of glucuronides (42.9% for genistein glucuronide (GG), 21.1% for apigenin glucuronide (AG) , and 33.7% for emodin glucuronide (EG); p < 0.01) and in cellular glucuronidation (38.3% for genistein, 38.6% for apigenin, and 34.7% for emodin; p < 0.01). Knock-down of a MRP transporter led to substantial decreases in excretion of GG (32.3% for MRP1, 36.7% for MRP3, and 36.6% for MRP4; p < 0.01) and AG (59.3% for MRP1, 24.7% for MRP3, and 34.1% for MRP4; p < 0.01). Also, cellular glucuronidation of genistein (38.3% for MRP1, 32.3% for MRP3, and 31.1% for MRP4; p < 0.01) and apigenin (40.6% for MRP1, 32.4% for MRP3, and 34.6% for MRP4; p < 0.001) was markedly suppressed. By contrast, silencing of MRPs did not cause any changes in either excretion of EG or cellular glucuronidation of emodin. In conclusion, cellular glucuronidation was significantly altered by decreasing expression of efflux transporters, revealing a strong interplay of glucuronidation with efflux transport.

Nanostructured lipid carriers used for oral delivery of oridonin: An effect of ligand modification on absorption

Oridonin (Ori) is a natural compound with notable anti-inflammation and anti-cancer activities. However, therapeutic use of this compound is limited by its poor solubility and low bioavailability. Here a novel biotin-modified nanostructured lipid carrier (NLC) was developed to enhance the bioavailability of Ori. The effect of ligand (biotin) modification on oral absorption of Ori encapsulated in NLCs was also explored. Ori-loaded NLCs (Ori-NLCs) were prepared by the melt dispersion-high pressure homogenization method. Biotin modification of Ori-NLCs was achieved by EDC and NHS in aqueous phase. The obtained biotin-decorated Ori-NLCs (Bio-Ori-NLCs) were 144.9nm in size with an entrapment efficiency of 49.54% and a drug load of 4.81%. Oral bioavailability was enhanced by use of Bio-Ori-NLCs with a relative bioavailability of 171.01%, while the value of non-modified Ori-NLCs was improved to 143.48%. Intestinal perfusion showed that Ori solution unexpectedly exhibited a moderate permeability, indicating that permeability was not a limiting factor of Ori absorption. Ori could be rapidly metabolized that was the main cause of low bioavailability. However, there was a difference in the enhancement of bioavailability between Bio-Ori-NLCs and conventional NLCs. Although severe lipolyses happened both on Bio-Ori-NLCs and non-modified NLCs, the performance of Bio-Ori-NLCs in the bioavailability improvement was more significant. Overall, Bio-Ori-NLCs can further promote the oral absorption of Ori by a ligand-mediated active transport. It may be a promising carrier for the oral delivery of Ori.

Quantitative analysis of Cistanches Herba using high‐performance liquid chromatography coupled with diode array detection and high‐resolution mass spectrometry combined with chemometric methods

We aim to determine the chemical constituents of three species of Cistanches Herba using HPLC coupled with diode array detection and high-resolution MS. Ten phenylethanoid glycosides were identified and further quantified as marker substances by HPLC coupled with diode array detection method. The separation was conducted using an Agilent TC-C18 column with 0.1% formic acid and methanol as the mobile phases under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and subsequently applied to evaluate the quality of 36 batches of Cistanche plants. The chemometric procedures (i.e., hierarchical clustering analysis and principal component analysis) were used to compare different species of Cistanches Herba, leading to successful classification of the Cistanche samples in accordance with their origins. In conclusion, this study provides a chemical basis for quality control of Cistanches Herba.

Effects of PEGylated lipid nanoparticles on the oral absorption of one BCS II drug: a mechanistic investigation

Lipid nanocarriers are becoming a versatile platform for oral delivery of lipophilic drugs. In this article, we aimed to explore the gastrointestinal behaviors of lipid nanoparticles and the effect of PEGylation on oral absorption of fenofibrate (FN), a Biopharmaceutics Classification System (BCS) II model drug. FN-loaded PEGylated lipid nanoparticles (FN-PLNs) were prepared by the solvent-diffusion method and characterized by particle size distribution, morphology, Fourier transform infrared spectroscopy, and drug release. Lipolytic experiments were performed to assess the resistance of lipid nanoparticles against pancreatic lipase. Pharmacokinetics was evaluated in rats after oral administration of FN preparations. The obtained FN-PLNs were 186.7 nm in size with an entrapment efficiency of >95%. Compared to conventional lipid nanoparticles, PLNs exhibited slower drug release in the lipase-containing medium, strikingly reduced mucin binding, and suppressed lipolysis in vitro. Further, oral absorption of FN was significantly enhanced using PLNs with relative bioavailability of 123.9% and 157.0% to conventional lipid nanoparticles and a commercial formulation (Lipanthyl®), respectively. It was demonstrated that reduced mucin trapping, suppressed lipolysis, and/or improved mucosal permeability were responsible for increased oral absorption. These results facilitated a better understanding of the in vivo fate of lipid nanoparticles, and suggested the potential of PLNs as oral carriers of BCS II drugs.

Structure‐based drug design of catechol‐O‐methyltransferase inhibitors for CNS disorders

Catechol‐O‐methyltransferase (COMT) is of great importance in pharmacology because it catalyzes the metabolism (methylation) of endogenous and xenobiotic catechols. Moreover, inhibition of COMT is the drug target in the management of central nervous system (CNS) disorders such as Parkinsons disease due to its role in regulation of the dopamine level in the brain. The X‐ray crystal structures for COMT have been available since 1994. The active sites for cofactor and substrate/inhibitor binding are well resolved to an atomic level, providing valuable insights into the catalytic mechanisms as well as the role of magnesium ions in catalysis. Determination of how the substrates/inhibitors bind to the protein leads to a structure‐based approach that has resulted in potent and selective inhibitors. This review focuses on the design of two types of inhibitors (nitrocatechol‐type and bisubstrate inhibitors) for COMT using the protein structures.

Metabolism of the anthelmintic drug niclosamide by cytochrome P450 enzymes and UDP-glucuronosyltransferases: metabolite elucidation and main contributions from CYP1A2 and UGT1A1

Abstract 1. Niclosamide is an old anthelmintic drug that shows potential in fighting against cancers. Here, we characterized the metabolism of niclosamide by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM) and expressed enzymes. 2. NADPH-supplemented HLM (and liver microsomes from various animal species) generated one hydroxylated metabolite (M1) from niclosamide; and UDPGA-supplemented liver microsomes generated one mono-O-glucuronide (M2). The chemical structures of M1 (3-hydroxy niclosamide) and M2 (niclosamide-2-O-glucuronide) were determined through LC–MS/MS and/or NMR analyses. 3. Reaction phenotyping revealed that CYP1A2 was the main enzyme responsible for M1 formation. The important role of CYP1A2 in niclosamide metabolism was further confirmed by activity correlation analyses as well as inhibition experiments using specific inhibitors. 4. Although seven UGT enzymes were able to catalyze glucuronidation of niclosamide, UGT1A1 and 1A3 were the enzymes showed the highest metabolic activities. Activity correlation analyses demonstrated that UGT1A1 played a predominant role in hepatic glucuronidation of niclosamide, whereas the role of UGT1A3 was negligible. 5. In conclusion, niclosamide was subjected to efficient metabolic reactions hydroxylation and glucuronidation, wherein CYP1A2 and UGT1A1 were the main contributing enzymes, respectively.