Prediction of herb-drug interactions involving consumption of furanocoumarin-mixtures and cytochrome P450 1A2-mediated caffeine metabolism inhibition in humans

Herb-drug interactions (HDI) has become important due to the increasing popularity of natural health product consumption worldwide. HDI is difficult to predict as botanical drugs usually contain complex phytochemical-mixtures, which interact with drug metabolism. Currently, there is no specific pharmacological tool to predict HDI since almost all in vitro-in vivo-extrapolation (IVIVE) Drug-Drug Interaction (DDI) models deal with one inhibitor-drug and one victim-drug. The objectives were to modify-two IVIVE models for the prediction of in vivo interaction between caffeine and furanocoumarin-containing herbs, and to confirm model predictions by comparing the DDI predictive results with actual human data. The models were modified to predict in vivo herb-caffeine interaction using the same set of inhibition constants but different integrated dose/concentration of furanocoumarin mixtures in the liver. Different hepatic inlet inhibitor concentration ([I]_H) surrogates were used for each furanocoumarin. In the first (hybrid) model, the [I]_H was predicted using the concentration-addition model for chemical-mixtures. In the second model, the [I]_H was calculated by adding individual furanocoumarins together. Once [I]_H values were determined, the models predicted an area-under-curve-ratio (AUCR) value of each interaction. The results indicate that both models were able to predict the experimental AUCR of herbal products reasonably well. The DDI model approaches described in this study may be applicable to health supplements and functional foods also.     

Clinical efficacies,underlying mechanisms and molecular targets of Chinese medicines for diabetic nephropathy treatment and management

Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN.     

pH-dependent transport kinetics of the human organic anion-transporting polypeptide 1A2

Organic anion-transporting polypeptide (OATP) 1A2 is expressed on the apical sides of intestinal and renal epithelial cells and considered to be involved in the intestinal absorption and renal reabsorption of drugs. Although the transport activity of OATP1A2 is considered to be pH-dependent, the effects of pH on its kinetic parameters and on the potency of OATP1A2 inhibitors are yet to be elucidated. Some OATP are known to have multiple binding sites (MBS), but it remains unclear whether OATP1A2 has MBS. In the present study, we evaluated the influence of pH on the OATP1A2-mediated uptake of estrone 3-sulfate using OATP1A2-expressing HEK293 cells. The uptake of 0.3 μM estrone 3-sulfate by HEK293-OATP1A2 cells was pH-dependent. OATP1A2 exhibited bimodal saturation kinetics at pH 6.3 and 7.4. Compared with that seen at pH 6.3 (5.62 μM), the K_m value of the high-affinity site was 8-fold higher at pH 7.4 (43.2 μM). In addition, the influence of pH on the potency of inhibitors varied among the examined inhibitors. These results suggest that the transport properties of OATP1A2 under lower pH conditions, such as those found in the microenvironments of the small intestinal mucosa and distal tubules, differ from those seen under neutral pH conditions.     

Characterization of organic anion transporting polypeptide 1b2 knockout rats generated by CRISPR/Cas9: a novel model for drug transport and hyperbilirubinemia disease

Organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1/3) as important uptake transporters play a fundamental role in the transportation of exogenous drugs and endogenous substances into cells. Rat OATP1B2, encoded by the Slco1b2 gene, is homologous to human OATP1B1/3. Although OATP1B1/3 is very important, few animal models can be used to study its properties. In this report, we successfully constructed the Slco1b2 knockout (KO) rat model via using the CRISPR/Cas9 technology for the first time. The novel rat model showed the absence of OATP1B2 protein expression, with no off-target effects as well as compensatory regulation of other transporters. Further pharmacokinetic study of pitavastatin, a typical substrate of OATP1B2, confirmed the OATP1B2 function was absent. Since bilirubin and bile acids are the substrates of OATP1B2, the contents of total bilirubin, direct bilirubin, indirect bilirubin, and total bile acids in serum are significantly higher in Slco1b2 KO rats than the data of wild-type rats. These results are consistent with the symptoms caused by the absence of OATP1B1/3 in Rotor syndrome. Therefore, this rat model is not only a powerful tool for the study of OATP1B2-mediated drug transportation, but also a good disease model to study hyperbilirubinemia-related diseases.     

A review of existing strategies for designing long-acting parenteral formulations: Focus on underlying mechanisms,and future perspectives

The need for long-term treatments of chronic diseases has motivated the widespread development of long-acting parenteral formulations (LAPFs) with the aim of improving drug pharmacokinetics and therapeutic efficacy. LAPFs have been proven to extend the half-life of therapeutics, as well as to improve patient adherence; consequently, this enhances the outcome of therapy positively. Over past decades, considerable progress has been made in designing effective LAPFs in both preclinical and clinical settings. Here we review the latest advances of LAPFs in preclinical and clinical stages, focusing on the strategies and underlying mechanisms for achieving long acting. Existing strategies are classified into manipulation of in vivo clearance and manipulation of drug release from delivery systems, respectively. And the current challenges and prospects of each strategy are discussed. In addition, we also briefly discuss the design principles of LAPFs and provide future perspectives of the rational design of more effective LAPFs for their further clinical translation.     

Allosteric inhibition reveals SHP2-mediated tumor immunosuppression in colon cancer by single-cell transcriptomics

Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING–TBK1–IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68⁺ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy.     

Bioengineered miR-328-3p modulates GLUT1-mediated glucose uptake and metabolism to exert synergistic antiproliferative effects with chemotherapeutics

MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/SLC2A1), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/SLC7A5) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism.     

Functional characterization of human organic anion transporter 10 (OAT10/SLC22A13) as an orotate transporter

Orotate, a nutritional compound typically utilized as an intermediate in pyrimidine synthesis, has been suggested to undergo renal reabsorption. However, the detailed mechanisms involved in the process remain unclear, with only urate transporter 1 (URAT1/SLC22A12) being indicated as a transporter involved in its tubular uptake. As an attempt to identify transporters involved in that to help clarify the mechanisms, we examined a possibility that organic anion transporter 10 (OAT10/SLC22A13), which is present at the brush border membrane in renal tubular epithelial cells, could transport orotate. The operation of human OAT10 for orotate transport was demonstrated indeed and analyzed in detail in Madin-Darby canine kidney II cells introduced with this transporter by stable transfection. Orotate transport by OAT10 was found to be kinetically saturable with a biphasic characteristic and dependent on Cl⁻. These are unique characteristics previously unknown in its operation for the other substrates. Orotate transport by OAT10 was, on the other hand, inhibited by several anionic compounds known as OAT10 inhibitors. Finally, the rat ortholog of OAT10 was found not to be able to transport orotate, indicating animal species differences in that function. Thus, human OAT10 has been demonstrated to operate for orotate transport with unique characteristics.     

Reconstitution of CYP3A4 active site through assembly of ligand interactions as a grid-template: Solving the modes of the metabolism and inhibition

A hexagonal-grid based template system has been developed to a predicting tool of CYP3A4-mediated reactions through the reconstitution of the active site with the assembly of the ligands. Simultaneous interactions of flattened-shape ligands at two sites of CYP3A4, oxidizing- and triggering-sites, are essential ideas, which were supported in the simulation results of various ligands on the template. The interactions were accomplished with either uni-molecule bindings or bi-molecule bindings with ligands termed pro-metabolized and trigger molecules. The template shape was determined mainly through reciprocal comparisons of simulation results with available experiment data mainly on recombinant CYP3A4-mediated reactions of polyaromatic hydrocarbon (PAH) ligands. Through the applications of various PAH and non-PAH ligands on the template, couple region-specific interactions including mechanisms to facilitate ligand movement from the initial site to a place near heme-oxygen and to trigger of catalyses are envisioned. These are very effective tools to verify candidates of CYP3A4 ligands as the good or poor substrates. The bi-molecule binding idea also explains so called “cooperative bindings with activator/effector” as interactions with non-identical trigger molecules on this CYP3A4-template system, and also offers possible mechanisms of the non-linear kinetic behavior.     

Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5

Programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blocking therapy has become a major pillar of cancer immunotherapy. Compared with antibodies targeting, small-molecule checkpoint inhibitors which have favorable pharmacokinetics are urgently needed. Here we identified berberine (BBR), a proven anti-inflammation drug, as a negative regulator of PD-L1 from a set of traditional Chinese medicine (TCM) chemical monomers. BBR enhanced the sensitivity of tumour cells to co-cultured T-cells by decreasing the level of PD-L1 in cancer cells. In addition, BBR exerted its antitumor effect in Lewis tumor xenograft mice through enhancing tumor-infiltrating T-cell immunity and attenuating the activation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). BBR triggered PD-L1 degradation through ubiquitin (Ub)/proteasome-dependent pathway. Remarkably, BBR selectively bound to the glutamic acid 76 of constitutive photomorphogenic-9 signalosome 5 (CSN5) and inhibited PD-1/PD-L1 axis through its deubiquitination activity, resulting in ubiquitination and degradation of PD-L1. Our data reveals a previously unrecognized antitumor mechanism of BBR, suggesting BBR is small-molecule immune checkpoint inhibitor for cancer treatment.     

Targeting human MutT homolog 1 (MTH1) for cancer eradication: current progress and perspectives

Since accelerated metabolism produces much higher levels of reactive oxygen species (ROS) in cancer cells compared to ROS levels found in normal cells, human MutT homolog 1 (MTH1), which sanitizes oxidized nucleotide pools, was recently demonstrated to be crucial for the survival of cancer cells, but not required for the proliferation of normal cells. Therefore, dozens of MTH1 inhibitors have been developed with the aim of suppressing cancer growth by accumulating oxidative damage in cancer cells. While several inhibitors were indeed confirmed to be effective, some inhibitors failed to kill cancer cells, complicating MTH1 as a viable target for cancer eradication. In this review, we summarize the current status of developing MTH1 inhibitors as drug candidates, classify the MTH1 inhibitors based on their structures, and offer our perspectives toward the therapeutic potential against cancer through the targeting of MTH1.     

Exploration of 5-cyano-6-phenylpyrimidin derivatives containing an 1,2,3-triazole moiety as potent FAD-based LSD1 inhibitors

Histone lysine specific demethylase 1 (LSD1) has become a potential therapeutic target for the treatment of cancer. Discovery and develop novel and potent LSD1 inhibitors is a challenge, although several of them have already entered into clinical trials. Herein, for the first time, we reported the discovery of a series of 5-cyano-6-phenylpyrimidine derivatives as LSD1 inhibitors using flavin adenine dinucleotide (FAD) similarity-based designing strategy, of which compound 14q was finally identified to repress LSD1 with IC₅₀ = 183 nmol/L. Docking analysis suggested that compound 14q fitted well into the FAD-binding pocket. Further mechanism studies showed that compound 14q may inhibit LSD1 activity competitively by occupying the FAD binding sites of LSD1 and inhibit cell migration and invasion by reversing epithelial to mesenchymal transition (EMT). Overall, these findings showed that compound 14q is a suitable candidate for further development of novel FAD similarity-based LSD1 inhibitors.     

Interacting with α7 nAChR is a new mechanism for AChE to enhance the inflammatory response in macrophages

Acetylcholine (ACh) regulates inflammation via α7 nicotinic acetylcholine receptor (α7 nAChR). Acetylcholinesterase (AChE), an enzyme hydrolyzing ACh, is expressed in immune cells suggesting non-classical function in inflammatory responses. Here, the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages. In lipopolysaccharide-induced inflammatory processes, AChE was upregulated by the binding of NF-κB onto the ACHE promotor. Conversely, the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration, which was in contrast to that of applied AChE inhibitors. AChEmt, a DNA construct without enzymatic activity, was adopted to identify the protein role of AChE in immune system. Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration. The co-localization of α7 nAChR and AChE was found in macrophages, suggesting the potential interaction of α7 nAChR and AChE. Besides, immunoprecipitation showed a close association of α7 nAChR and AChE protein in cell membrane. Hence, the novel function of AChE in macrophage by interacting with α7 nAChR was determined. Together with hydrolysis of ACh, AChE plays a direct role in the regulation of inflammatory response. As such, AChE could serve as a novel target to treat age-related diseases by anti-inflammatory responses.     

Impact of molecular weight on the mechanism of cellular uptake of polyethylene glycols (PEGs) with particular reference to P-glycoprotein

Polyethylene glycols (PEGs) in general use are polydisperse molecules with molecular weight (MW) distributed around an average value applied in their designation e.g., PEG 4000. Previous research has shown that PEGs can act as P-glycoprotein (P-gp) inhibitors with the potential to affect the absorption and efflux of concomitantly administered drugs. However, questions related to the mechanism of cellular uptake of PEGs and the exact role played by P-gp has not been addressed. In this study, we examined the mechanism of uptake of PEGs by MDCK-mock cells, in particular, the effect of MW and interaction with P-gp by MDCK-hMDR1 and A549 cells. The results show that: (a) the uptake of PEGs by MDCK-hMDR1 cells is enhanced by P-gp inhibitors; (b) PEGs stimulate P-gp ATPase activity but to a much lesser extent than verapamil; and (c) uptake of PEGs of low MW (<2000 Da) occurs by passive diffusion whereas uptake of PEGs of high MW (>5000 Da) occurs by a combination of passive diffusion and caveolae-mediated endocytosis. These findings suggest that PEGs can engage in P-gp-based drug interactions which we believe should be taken into account when using PEGs as excipients and in PEGylated drugs and drug delivery systems.     

Synergistic antitumor activity of artesunate and HDAC inhibitors through elevating heme synthesis via synergistic upregulation of ALAS1 expression

Artemisinin and its derivatives (ARTs) were reported to display heme-dependent antitumor activity. On the other hand, histone deacetylase inhibitors (HDACi) were known to be able to promote heme synthesis in erythroid cells. Nevertheless, the effect of HDACi on heme homeostasis in non-erythrocytes remains unknown. We envisioned that the combination of HDACi and artesunate (ARS) might have synergistic antitumor activity through modulating heme synthesis. In vitro studies revealed that combination of ARS and HDACi exerted synergistic tumor inhibition by inducing cell death. Moreover, this combination exhibited more effective antitumor activity than either ARS or HDACi monotherapy in xenograft models without apparent toxicity. Importantly, mechanistic studies revealed that HDACi coordinated with ARS to increase 5-aminolevulinate synthase (ALAS1) expression, and subsequent heme production, leading to enhanced cytotoxicity of ARS. Notably, knocking down ALAS1 significantly blunted the synergistic effect of ARS and HDACi on tumor inhibition, indicating a critical role of ALAS1 upregulation in mediating ARS cytotoxicity. Collectively, our study revealed the mechanism of synergistic antitumor action of ARS and HDACi. This finding indicates that modulation of heme synthesis pathway by the combination based on ARTs and other heme synthesis modulators represents a promising therapeutic approach to solid tumors.