Effect of a new inhibitor of factor Xa zifaxaban, on thrombosis in the inferior vena cava in rabbits.

In recent years, oral factor Xa inhibitors have become a research focus as anticoagulant drugs. Zifaxaban is the first oral FXa inhibitor to enter clinical trials in China. The aim of this study was to determine the inhibitory effect of zifaxaban on thrombosisthrough a model ofinferior vena cava (IVC) thrombosis in rabbits. IVC thrombosis model was established by electrical injury and stenosis, and zifaxaban was administered (p.o.) for 5 consecutive days, then coagulation indicators and bleeding were observed. The results showed that zifaxaban had obvious inhibitory effects on FXa, and had a significant inhibitory effect on IVC thrombosis induced by electrical damage and stenosis. The effect of zifaxaban was similar to that of rivaroxaban, but the bleeding side-effects of zifaxaban were less severe than those of rivaroxaban. Zifaxaban could prolong the prothrombin time and activated partial thromboplastin time of plasma similar to that of other oral FXa inhibitors. Zifaxaban had a significant inhibitory effect on FXa, but it had no obvious effect on other coagulation factors, major anticoagulant factors or fibrinolytic indices. Our results suggest that zifaxaban had specific inhibitory effects on FXa and inhibited IVC thrombosis in rabbits with its hemorrhagic effect was less than that of rivaroxaban. Zifaxaban is ecpected to be developed as a new drug for the prevention of deep venous thrombosis, providing more medication options for patients with such disease, more research is required to support it in the future.

Systematic Structure-Activity Relationship (SAR) Exploration of Diarylmethane Backbone and Discovery of A Highly Potent Novel Uric Acid Transporter 1 (URAT1) Inhibitor.

In order to systematically explore and better understand the structure-activity relationship (SAR) of a diarylmethane backbone in the design of potent uric acid transporter 1 (URAT1) inhibitors, 33 compounds (1a-1x and 1ha-1hi) were designed and synthesized, and their in vitro URAT1 inhibitory activities (IC50) were determined. The three-round systematic SAR exploration led to the discovery of a highly potent novel URAT1 inhibitor, 1h, which was 200- and 8-fold more potent than parent lesinurad and benzbromarone, respectively (IC50 = 0.035 µM against human URAT1 for 1h vs. 7.18 µM and 0.28 µM for lesinurad and benzbromarone, respectively). Compound 1h is the most potent URAT1 inhibitor discovered in our laboratories so far and also comparable to the most potent ones currently under development in clinical trials. The present study demonstrates that the diarylmethane backbone represents a very promising molecular scaffold for the design of potent URAT1 inhibitors.

Discovery of a Flexible Triazolylbutanoic Acid as a Highly Potent Uric Acid Transporter 1 (URAT1) Inhibitor.

In order to systematically explore and understand the structure-activity relationship (SAR) of a lesinurad-based hit (1c) derived from the replacement of the S atom in lesinurad with CH2, 18 compounds (1a-1r) were designed, synthesized and subjected to in vitro URAT1 inhibitory assay. The SAR exploration led to the discovery of a highly potent flexible URAT1 inhibitor, 1q, which was 31-fold more potent than parent lesinurad (IC50 = 0.23 µM against human URAT1 for 1q vs 7.18 µM for lesinurad). The present study discovered a flexible molecular scaffold, as represented by 1q, which might serve as a promising prototype scaffold for further development of potent URAT1 inhibitors, and also demonstrated that the S atom in lesinurad was not indispensable for its URAT1 inhibitory activity.

Design, synthesis and antitumor activity of 4-aminoquinazoline derivatives targeting VEGFR-2 tyrosine kinase.

We report herein the design and synthesis of novel 4-aminoquinazoline derivatives based on the inhibitors of VEGFR-2 tyrosine kinases. The VEGFR-2 inhibitory activities of these newly synthesized compounds were also evaluated and compared with that of ZD6474. We found that most of target compounds had good inhibitory potency. In particular, compounds 1h, 1n and 1o were found to be 6, 2 and 2-fold more potent than the positive control ZD6474. The leading compound 1h also showed an in vivo activity against HepG2 human tumor xenograft model in BALB/c-nu mice.

(4-Meth-oxy-phen-yl)(4-methyl-cyclo-hex-yl)methanone.

The title compound, C(15)H(20)O(2), crystallizes with two independent mol-ecules of similar geometry in the asymmetric unit. The cyclo-hexyl ring adopts a chair conformation in each mol-ecule. In the crystal, mol-ecules related by translation are linked into chains along the a axis via weak C-H⋯O inter-actions.

Corrigendum to "Effects of total iridoid glycosides of Picrorhiza scrophulariiflora against non-alcoholic steatohepatitis rats induced by high-fat and high-sugar diet through regulation of lipid metabolism" [Chin Herb Med 12 (2019) 67-72].

[This corrects the article DOI: 10.1016/j.chmed.2019.12.005.].

Corrigendum to "Neuroprotective effect of Sanqi Tongshuan Tablets on sequelae post-stroke in rats" [Chinese Herbal Medicines 11 (2019) 45-51].

[This corrects the article DOI: 10.1016/j.chmed.2018.12.002.].

Improved Antiglioblastoma Activity and BBB Permeability by Conjugation of Paclitaxel to a Cell-Penetrative MMP-2-Cleavable Peptide.

In order to solve the problems of receptor promiscuity and poor blood-brain barrier (BBB) penetration in the treatment of glioblastomas (GBM), a novel dual-functional nanocomplex drug delivery system is developed based on the strategy of peptide-drug conjugates. In this study, SynB3-PVGLIG-PTX is designed and screened out by matrix metalloproteinase-2 (MMP-2), to which it exhibits the best affinity. The MMP-2-sensitive peptide (PVGLIG) and a cell-penetration peptide (SynB3) are combined to form a dual-functional peptide. Moreover, as a drug-peptide nanocomplex, SynB3-PVGLIG-PTX exhibited a high potential to form an aggregation with good solubility that can release paclitaxel (PTX) through the cleavage of MMP-2. From a functional perspective, it is found that SynB3-PVGLIG-PTX can specifically inhibit the proliferation, migration, and invasion of GBM cells in vitro in the presence of MMP-2, in contrast to that observed in MMP-2 siRNA transfected cells. Further investigation in vivo shows that SynB3-PVGLIG-PTX easily enters the brain of U87MG xenograft nude mice and can generate a better suppressive effect on GBM through a controlled release of PTX from SynB3-PVGLIG-PTX compared with PTX and temozolomide. Thus, it is proposed that SynB3-PVGLIG-PTX can be used as a novel drug-loading delivery system to treat GBM due to its specificity and BBB permeability.

Effects of total iridoid glycosides of Picrorhiza scrophulariiflora against non-alcoholic steatohepatitis rats induced by high-fat and high-sugar diet through regulation of lipid metabolism.

Objective: To investigate the therapeutic effect of total iridoid glycosides of Picrorhiza scrophulariiflora (TIGP) on non-alcoholic steatohepatitis (NASH). Methods: SD rats were fed with high-fat and high-sugar diet for 8 weeks to establish NASH. TIGP were given orally at doses of 20, 40 and 80 mg/kg/d for 4 weeks. Triglycerides assay (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), fasting plasma glucose (FPG), fasting insulin (FINS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), chemokine-1 (MCP-1), leptin (LEP) in serum were tested. TG, TC, superoxide dismutase (SOD), malondialdehyde (MDA), and free fatty acid (FFA) in liver tissue were determined by colorimetric methods. Steatosis of hepatocytes and inflammation was performed by pathological examination. Results: The results showed that TIGP significantly decreased TC, TG and FFA in liver tissue, increased SOD activity, decreased MDA content, decreased serum levels of TG, TC, HDL-C/LDL-C, ALT, AST, GLU, HOMA-IR, TNF-α and LEP, and in addition, improved steatosis of liver cells compared to NASH. Conclusion: TIGP had anti-fatty liver effect against NASH rats induced by high-fat and high-sugar diet. Its mechanism was related to the regulation of lipid metabolism and reduction of insulin resistance, through inhibition of oxidative stress and inflammation.

Penetration enhancement of menthol on quercetin through skin: insights from atomistic simulation.

Menthol is an often used skin penetration enhancer because of its high efficiency and relative safety, but the mechanism how it works has not been fully understood up to date. In this study, quercetin was used as a model molecule to investigate the permeability enhancement of menthol through skin lipids. The skin is modeled as a ceramide (CER2) bilayer. Potential of mean force (PMF) calculations on quercetin in both CER2 and menthol-involved CER2 bilayers have been performed. The results show that the free energy minimum of quercetin in the presence of menthol molecules shifts toward the headgroup region of the bilayer, and the central energy barrier decreases, facilitating the penetration of quercetin. The presence of menthol molecules enhances the permeability of quercetin. This study may shed light on the mechanism of penetration enhancer, providing useful information in the design of more efficient transdermal drug delivery system. Graphical abstract Quercetin was used as a model molecule to investigate the permeability enhancement of menthol through skin lipids. Potential of mean force calculations reveal that the central energy barrier of quercetin decreases in the presence of menthol, facilitating the penetration of quercetin. Our results are helpful to understand the mechanism of penetration enhancer, aiding in the design of more efficient transdermal drug delivery system.

Thrombolysis with rhPro-UK 3 to 6 hours after embolic stroke in rat.

Objectives: To investigate the thrombolysis with recombinant human prourokinase (rhPro-UK) on thromboembolic stroke in rats at different therapeutic time windows (TTW). Methods: Rats were subjected to embolic middle cerebral artery occlusion. RhPro-UK and positive control drugs rt-PA,UK were administered 3 h, 4.5 h, 6 h after inducing thromboem-bolic stroke. Neurological deficit scoring (NDS) was evaluated at 6 h and 24 h after the treatment. The lesion volume in cerebral hemispheres was measured by MRI scanning machine after 6 h of thrombolysis, and the infarct volume was measured by TTC stain, together with hemorrhagic volume quantified by a spectrophotometric assay after 24 h of thrombolysis. Results: RhPro-UK 10, 20 × 104 U/kg significantly improved the NDS after cerebral thromboembolism in rats at 3 h, 4.5 h TTW, and at the 6 h TTW, the NDS was improved by 28.0% (P = 0.0690) and 29.2% (P = 0.0927) at 6 h and 24 h after rhPro-UK 20 ×104 U/kg administration, respectively. RhPro-UK 10, 20 × 104 U/kg significantly reduced the brain lesions measured by MRI at 3 h and 4.5 h TTW. RhPro-UK 10, 20 × 104 U/kg significantly reduced the cerebral infarction measured by TTC at 3 h, 4.5 h TTW. There was no increase in cerebral hemorrhage compared with untreated group after rhPro-UK administration. Conclusions: RhPro-UK had an obvious therapeutic effect on ischemic stroke caused by thrombosis, and could be started within 4.5 h TTW with less side effects of cerebral hemorrhage than that of UK.

1-(4-Methoxy-phen-yl)-3-methyl-1H-1,2,4-triazol-5(4H)-one.

In the title compound, C(10)H(11)N(3)O(2), the triazole ring has a dihedral angle of 11.55 (2)° relative to the benzene ring. The crystal packing is stabilized by inter-molecular N-H⋯O and C-H⋯O hydrogen bonds, and by weak π-π stacking inter-actions [centroid-to-centroid distance = 3.545 (3) Å].

Pre-clinical pharmacodynamic study of a novel oral factor Xa inhibitor zifaxaban.

Zifaxaban is an orally active, direct Factor Xa (FXa) inhibitor that is in development for the prevention and treatment of arterial and venous thrombosis. This study was conducted to investigate the biochemical and pharmacological activity of zifaxaban. In vitro activity was evaluated by enzyme, platelet aggregation, and clotting assays. In vivo effects were examined in venous thrombosis, arteriovenous-shunt thrombosis, carotid thrombosis, and bleeding models in rats. Zifaxaban competitively inhibits human FXa (IC50 = 11.1 nM) with > 10,000-fold greater selectivity than other serine proteases. It did not impair platelet aggregation induced by collagen, adenosine diphosphate (ADP) or arachidonic acid. It significantly prolonged clotting time, prothrombin time (PT), and activated partial thromboplastin time (APTT) in the plasma of humans, rabbits, and rats, with a relatively weak effect on thrombin time (TT). In venous thrombosis models in rats, zifaxaban strongly suppressed thrombus formation with ED50 values of 3.09 mg/kg, and its best efficacy time occurred at 2 h after administration. In arteriovenous-shunt thrombosis and carotid thrombosis models in rats, it inhibited thrombus formation in a dose-dependent manner. And in the rat tail bleeding assay, it showed a trend of less bleeding than rivaroxaban at doses that achieved the same antithrombotic effect. In conclusion, zifaxaban is a selective and direct FXa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.

Effect of recombinant human prourokinase on thrombolysis in a rabbit model of thromboembolic stroke.

The aim of the present study was to investigate the efficacy of recombinant human prourokinase (rhPro-UK) on thromboembolic stroke in rabbits. A total of 210 rabbits were used in experiments. The 180 thromboembolic stroke rabbits were divided into three therapeutic time windows with six groups in each time window (n=10). The model group was administered saline, the reagent groups were administered rhPro-UK (2.5×, 5× and 10×104 U/kg), and the positive control groups were administered 5×104 urokinase (UK) U/kg and 4.5 mg/kg recombinant human tissue plasminogen activator via intravenous infusion at 3, 4.5 and 6 h after embolism. The remaining 30 rats (that had not undergone occlusion by autologous blood clots) served as a sham group and were administered saline. The radioactive intensity was detected using a medical gamma counter before and after the administration of the drug for 15, 30, 45, 60, 75, 90, 105 and 120 min. At 24 h after treatment, the brain samples were coronally sliced into 5 mm sections and hemorrhage was estimated used a semiquantitative method by counting the number of section faces with hemorrhaging. The plasma was collected for prothrombin time, activated partial thromboplastin time, fibrinogen and thrombin time tests using a solidification method with a blood coagulation factor analyzer. In addition, α2-antiplasmin (α2-AP) was evaluated using ELISA methods using a RT-6100 microplate reader. At the 3 h time point, the thrombolysis rate of rhPro-UK(2.5×, 5× and 10×104 U/kg) was 21.5% (P<0.05), 36.8% (P<0.001) and 55.0% (P<0.001), respectively together with patency rates of 10% (P>0.05), 40% (P<0.05) and 70% (P<0.001). Furthermore, α2-AP levels were reduced by 5.3% (P>0.05), 5.3% (P>0.05) and 18.1% (P<0.05). At the 4.5 h time point, the thrombolysis rate was 18.8% (P<0.05), 29.9% (P<0.01) and 49.0% (P<0.001) together with patency rates of 10% (P>0.05), 30% (P<0.05) and 50% (P<0.01), and α2-AP levels were reduced by 2.4% (P>0.05), 6.5% (P>0.05) and 17.8% (P<0.05). At the 6 h time point, the thrombolysis rate was 14.7% (P<0.05), 24.1%(P<0.01) and 35.7% (P<0.001) together with patency rates of 20% (P>0.05), 30% (P<0.05) and 40% (P<0.01), and α2-AP levels were reduced by 5.7% (P>0.05), 12.7% (P>0.05) and 22.2% (P<0.01). No significant differences (P>0.05) were identified between rhPro-UK (2.5×, 5× and 10×104 U/kg) and the model group regarding hemorrhage type, size and blood coagulation factors at the different time points. Thus, rhPro-UK promoted thrombolysis and recanalization (patency rate), with reduced risk of cerebral hemorrhage, and thus exerted protective effects on cerebral ischemia rabbits.

Effect of human recombinant prourokinase(rhpro-UK) on thromboembolic stroke in rats.

We evaluated the efficacy and safety of human recombinant prourokinase ( rhpro-UK) on thromboembolic stroke in rats. 60 rats with thromboembolic stroke were divided into 6 groups (n = 10). The model group was given saline, the reagent groups were given rhpro-UK (5, 10, 20 × 104U/kg), and positive control groups were given urokinase (UK) 10 × 104U/kg and recombinant tissue plasminogen activator (rt-PA) 9mg/kg through intravenous infusion at 1.5h after embolism. And other 10 rats without occluded by autologous blood clots as the sham group were given saline. At 6h after treatment, neurological deficit score and Magnetic Resonance Imaging(MRI) including T1WI and T2WI sequence scanning were measured. At 24h after treatment, the brain was cut for 2,3,5-triphenyltetrazolium chloride (TTC) staining and aspectrophotometric assay to measure the infarct area and intracerebral hemorrhage after neurological deficit detection. rhpro-UK (5, 10, 20 × 104 U/kg) improved neurological disorder by 39.1 ± 19.7% (n = 10, P > 0.05), 65.2 ± 14.2% (n = 10, P < 0.01) and 65.2 ± 14.2% (n = 10, P < 0.01) maximally; decreased brain lesion volume by 36.7 ± 34.8% (n = 10, P < 0.05), 77.6 ± 7.7% (n = 10, P < 0.01) and 80.5 ± 6.9% (n = 10, P < 0.01); decreased infarction area by 38.2 ± 24.0% (n = 10, P < 0.01), 73.9 ± 5.2% (n = 10, P < 0.001) and 79.7 ± 4.0% (n = 10, P < 0.001) respectively, and there were no statistics difference between rhpro-UK (5, 10, 20 × 104 U/kg) and each positive groups at intracerebral hemorrhage (P > 0.05). Rhpro-UK improved the damaged neural function, decreased the extent of the disease and did not raise bleeding, had protective effects for cerebral ischemia in rats.

Discovery of Flexible Naphthyltriazolylmethane-based Thioacetic Acids as Highly Active Uric Acid Transporter 1 (URAT1) Inhibitors for the Treatment of Hyperuricemia of Gout.

BACKGROUND: Gout is the most common inflammatory arthritis, which, if left untreated or inadequately treated, will lead to joint destruction, bone erosion and disability due to the crystal deposition. Uric acid transporter 1 (URAT1) was the promising therapeutic target for urate-lowering therapy. OBJECTIVE: The goal of this work is to understand the structure-activity relationship (SAR) of a potent lesinurad-based hit, sodium 2-((5-bromo-4-((4-cyclopropyl-naphth-1-yl)methyl)-4H-1,2,4-triazol-3- yl)thio)acetate (1c), and based on that discover a more potent URAT1 inhibitor. METHODS: The SAR of 1c was systematically explored and the in vitro URAT1 inhibitory activity of synthesized compounds 1a-1t was determined by the inhibition of URAT1-mediated [8-14C]uric acid uptake by human embryonic kidney 293 (HEK293) cells stably expressing human URAT1. RESULTS: Twenty compounds 1a-1t were synthesized. SAR analysis was performed. Two highly active URAT1 inhibitors, sodium 2-((5-bromo-4-((4-n-propylnaphth-1-yl)methyl)-4H-1,2,4-triazol-3- yl)thio)acetate (1j) and sodium 2-((5-bromo-4-((4-bromonaphth-1-yl)methyl)-4H-1,2,4-triazol-3- yl)thio)acetate (1m), were identified, which were 78- and 76-fold more active than parent lesinurad in in vitro URAT1 inhibitory assay, respectively (IC50 values for 1j and 1m were 0.092 µM and 0.094 µM, respectively, against human URAT1 vs 7.18 µM for lesinurad). CONCLUSION: Two highly active URAT1 inhibitors were discovered. The SAR exploration also identified more flexible naphthyltriazolylmethane as a novel molecular skeleton that will be valuable for the design of URAT1 inhibitors, as indicated by the observation that many of the synthesized naphthyltriazolylmethane- bearing derivatives (1b-1d, 1g, 1j and 1m) showed significantly improved UART1 inhibitory activity (sub-micromolar IC50 values) as compared with lesinurad which has the rigid naphthyltriazole skeleton.

Variant fatty acid-like molecules Conjugation, novel approaches for extending the stability of therapeutic peptides.

The multiple physiological properties of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the treatment of type 2 diabetes. However, the in vivo half-life of GLP-1 is short due to rapid degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. The poor stability of GLP-1 has significantly limited its clinical utility; however, many studies are focused on extending its stability. Fatty acid conjugation is a traditional approach for extending the stability of therapeutic peptides because of the high binding affinity of human serum albumin for fatty acids. However, the conjugate requires a complex synthetic approach, usually involving Lys and occasionally involving a linker. In the current study, we conjugated the GLP-1 molecule with fatty acid derivatives to simplify the synthesis steps. Human serum albumin binding assays indicated that the retained carboxyl groups of the fatty acids helped maintain a tight affinity to HSA. The conjugation of fatty acid-like molecules improved the stability and increased the binding affinity of GLP-1 to HSA. The use of fatty acid-like molecules as conjugating components allowed variant conjugation positions and freed carboxyl groups for other potential uses. This may be a novel, long-acting strategy for the development of therapeutic peptides.

Discovery of 6-deoxydapagliflozin as a highly potent sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor for the treatment of type 2 diabetes.

Systematic mono-deoxylation of the four hydroxyl groups in the glucose moiety in dapagliflozin led to the discovery of 6-deoxydapagliflozin 1 as a more active sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor (IC50 = 0.67 nM against human SGLT2 (hSGLT2) vs 1.16 nM for dapagliflozin). It exhibited more potent blood glucose inhibitory activity in rat oral glucose tolerance test and induced more urinary glucose in rat urinary glucose excretion test than its parent compound dapagliflozin.

Potent tumor targeting drug release system comprising MMP-2 specific peptide fragment with self-assembling characteristics.

Self-assembling peptides are capable of forming a complex containing a cavity where cytotoxic agents can be wrapped in a self-assembling manner. These complexes are beneficial for improving the pharmacological properties and pharmacokinetics of cytotoxic agents, such as doxorubicin and paclitaxel. In the present study, this self-assembling feature was successfully integrated into a hexapeptide with matrix metalloproteinase (MMP)-2 specific targeting activity, producing a supramolecule possessing controlled drug release characteristics. The MMP-2 specific substrate fragment, PVGLIG, makes this supramolecule disassociate in the presence of MMP-2, and this system is considered to be a powerful tool for the treatment of tumors with high expression of MMP-2 or tumor metastasis. Our findings show that this modified self-assembling peptide with the PVGLIG fragment was able to significantly enhance specificity against HT1080 cells, a tumor cell line with high expression of MMP-2. In addition, residence time of the complex in blood was prolonged since paclitaxel was wrapped into the supramolecule. Our results suggest that the modified MMP-2 specific substrate, SAMTA7, could act as a controlled and sustained drug carrier for treatment of tumors with high expression of MMP-2 and for tumor metastasis.

Developments of glucagon like peptide-1 (GLP-1) and long-acting analogs in clinical and preclinical studies for treatment of type 2 diabetes.

The outstanding physiological functions of glucagon-like peptide-1 make it a promising drug candidate for blood glucose regulation in type 2 diabetes. However, the short half-life of GLP-1 limited its widely clinical utility due to the rapid degradation by dipeptidyl peptidase IV (DPP-IV) and renal clearance. Therefore, stabilisation of glucagon-like peptide-1 is critical for the use of this peptide in drug development. Scientists in pharmaceutical companies have contributed in years to obtain long-acting or sustained GLP-1 derivatives which are reviewed in this report.