Orally bioavailable amine-linked macrocyclic inhibitors of factor XIa

The discovery of orally bioavailable FXIa inhibitors has been a challenge. Herein, we describe our efforts to address this challenge by optimization of our imidazole-based macrocyclic series. Our optimization strategy focused on modifications to the P2 prime, macrocyclic amide linker, and the imidazole scaffold. Replacing the amide of the macrocyclic linker with amide isosteres led to the discovery of substituted amine linkers which not only maintained FXIa binding affinity but also improved oral exposure in rats. Combining the optimized macrocyclic amine linker with a pyridine scaffold afforded compounds 23 and 24 that were orally bioavailable, single-digit nanomolar FXIa inhibitors with excellent selectivity against relevant blood coagulation enzymes.     

Allosteric Inhibition as a New Mode of Action for BAY 1213790, a Neutralizing Antibody Targeting the Activated Form of Coagulation Factor XI

Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa–BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.     

Macrocyclic inhibitors of Factor XIa: Discovery of alkyl-substituted macrocyclic amide linkers with improved potency

Optimization of macrocyclic inhibitors of FXIa is described which focused on modifications to both the macrocyclic linker and the P1 group. Increases in potency were discovered through interactions with a key hydrophobic region near the S1 prime pocket by substitution of the macrocyclic linker with small alkyl groups. Both the position of substitution and the absolute stereochemistry of the alkyl groups on the macrocyclic linker which led to improved potency varied depending on the ring size of the macrocycle. Replacement of the chlorophenyltetrazole cinnamide P1 in these optimized macrocycles reduced the polar surface area and improved the oral bioavailability for the series, albeit at the cost of a decrease in potency.     

An anticoagulant peptide from the human hookworm, Ancylostoma duodenale that inhibits coagulation factors Xa and XIa

A full-length cDNA encoding an anticoagulant peptide, named AduNAP4, was cloned and identified from the human hookworm Ancylostoma duodenale. AduNAP4 has 104 amino acids including a predicted 23-residue signal peptide and shows ?50% similarity with other known nematode anticoagulant protein/peptide (NAP). AduNAP4 is extremely efficient at prolonging the activated partial thromboplastin time, and is an inhibitor of both fXa (K_i = 7.34 ± 1.74 nM) and fXIa (K_i = 42.45 ± 3.25 nM). No fXIa inhibitor has previously been described from other blood-feeding animals. Our results suggest that hookworms have evolved a potent mechanism that interferes with coagulation by inhibition of fXIa to facilitate its blood-feeding lifestyle.     

Combinatorial anticancer effects of curcumin and 5-fluorouracil loaded thiolated chitosan nanoparticles towards colon cancer treatment

Background

Evaluation of the combinatorial anticancer effects of curcumin/5-fluorouracil loaded thiolated chitosan nanoparticles (CRC-TCS-NPs/5-FU-TCS-NPs) on colon cancer cells and the analysis of pharmacokinetics and biodistribution of CRC-TCS-NPs/5-FU-TCS-NPs in a mouse model.

Methods

CRC-TCS-NPs/5-FU-TCS-NPs were developed by ionic cross-linking. The in vitro combinatorial anticancer effect of the nanomedicine was proven by different assays. Further the pharmacokinetics and biodistribution analyses were performed in Swiss Albino mouse using HPLC.

Results

The 5-FU-TCS-NPs (size: 150 ± 40 nm, zeta potential: + 48.2 ± 5 mV) and CRC-TCS-NPs (size: 150 ± 20 nm, zeta potential: + 35.7 ± 3 mV) were proven to be compatible with blood. The in vitro drug release studies at pH 4.5 and 7.4 showed a sustained release profile over a period of 4 days, where both the systems exhibited a higher release in acidic pH. The in vitro combinatorial anticancer effects in colon cancer (HT29) cells using MTT, live/dead, mitochondrial membrane potential and cell cycle analysis measurements confirmed the enhanced anticancer effects (2.5 to 3 fold). The pharmacokinetic studies confirmed the improved plasma concentrations of 5-FU and CRC up to 72 h, unlike bare CRC and 5-FU.

Conclusions

To conclude, the combination of 5-FU-TCS-NPs and CRC-TCS-NPs showed enhanced anticancer effects on colon cancer cells in vitro and improved the bioavailability of the drugs in vivo.

General significance

The enhanced anticancer effects of combinatorial nanomedicine are advantageous in terms of reduction in the dosage of 5-FU, thereby improving the chemotherapeutic efficacy and patient compliance of colorectal cancer cases.     

Development and characterization of lipidic cochleate containing recombinant factor VIII

Hemophilia A, a life-threatening bleeding disorder, is caused by deficiency of factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15-30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII-containing cochleate cylinders, utilizing PS-Ca²⁺ interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze-thawing and rate-limiting diffusion studies revealed that small cochleate cylinders with a particle size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII-cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications.     

Tick salivary secretion as a source of antihemostatics

Ticks are mostly obligatory blood feeding ectoparasites that have an impact on human and animal health. In addition to direct damage due to feeding, some tick species serve as the vectors for the causative agents of several diseases, such as the spirochetes of the genus Borrelia causing Lyme disease, the virus of tick-borne encephalitis, various Rickettsial pathogens or even protozoan parasites like Babesia spp. Hard ticks are unique among bloodfeeders because of their prolonged feeding period that may last up to two weeks. During such a long period of blood uptake, the host develops a wide range of mechanisms to prevent blood loss. The arthropod ectoparasite, in turn, secretes saliva in the sites of bite that assists blood feeding. Indeed, tick saliva represents a rich source of proteins with potent pharmacologic action that target different mechanisms of coagulation, platelet aggregation and vasoconstriction. Tick adaptation to their vertebrate hosts led to the inclusion of a powerful protein armamentarium in their salivary secretion that has been investigated by high-throughput methods. The resulting knowledge can be exploited for the isolation of novel antihemostatic agents. Here we review the tick salivary antihemostatics and their characterized functions at the molecular and cellular levels.     

Expression and characterization of wild-type TFPI and the [P151L]TFPI mutant in insect cells

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation. In earlier studies we could identify the [P151L]TFPI mutant, and we could also demonstrate that heterozygous carriers of this mutant show a nine-fold increased risk for deep venous thrombosis (DVT). To express greater amounts of both proteins and to enable their characterization, we expressed wild-type TFPI as well as [P151L]TFPI in High Five insect cells with expression rates of up to 215 ng/ml for wild-type TFPI and 214 ng/ml for [P151L]TFPI. The specific inhibitory activities for the recombinant proteins were determined as 11.3 and 11.5 mU/ng, respectively. Both proteins were detected via Western blot analysis and ELISA. The recombinant proteins' inhibitory activities were characterized by a chromogenic assay and by the determination of a modified activated thromboplastin time (aPTT) in which both of them proved to be inhibitorily active. We also examined both recombinant proteins' binding properties to glycoproteins, glycosaminoglycans, lipoproteins and tissue factor. Our results show that we have developed an efficient model system for the recombinant expression of inhibitorily active wild-type TFPI as well as [P151L]TFPI in insect cells, and we were able to characterize both proteins' inhibitory properties by determination of their influence on the aPTT and also their binding properties. Although both recombinant proteins did not show a significant difference in their effect on the aPTT, their binding properties differed significantly between the wild type and mutant protein.     

Anticoagulant activity of M-LAO, l-amino acid oxidase purified from Agkistrodon halys blomhoffii, through selective inhibition of factor IX

One of haemorrhagic toxins present in snake venoms is l-amino acid oxidase (LAO), which catalyzes the oxidative deamination of l-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.