Minimally Invasive Hemostatic Materials: Tackling a Dilemma of Fluidity and Adhesion by Photopolymerization in situ.

Hemostasis in vivo is a key to success in minimally invasive surgery (MIS). However, solid hemostatic materials cannot pass through the sheath tube of the MIS apparatus, while liquid ones are restricted by their low adhesion, which leads to them peeling off of tissue. To tackle the dilemma of fluidity and adhesion, a formulation containing a multifunctional sucrose allyl ether (SAE) monomer and an alpha-hydroxyketone liquid photoinitiator (HMPP) was applied as a lead hemostatic material for MIS. Real-time infrared results showed that SAE initiated by HMPP can rapidly polymerize into a transparent crosslinking membrane. Quantum chemistry showed that this occurs via a free radical addition reaction mechanism. Thermodynamic properties, such as reaction driving force and enthalpy change, were similar to those for a corresponding small molecular analogue, allyl methyl ether (AME), but the addition rate was lower than that for AME. The CC50 values of SAE and HMPP were also obtained by cell experiments. A hemostasis experiment in vivo was performed by comparing the formulation with chitosan and a traditional Chinese medicine (Yunnan Baiyao powder). The result showed that the formulation had a competitive advantage for use in MIS.

Half-life extended factor VIII for the treatment of hemophilia A.

Prophylactic infusion of factor VIII (FVIII) prevents joint bleeding and other hemorrhages in patients with hemophilia A. Conventional FVIII concentrates have a short half-life, with an average of about 12 h in adults, ranging in individual patients between 6 and 24 h, and even shorter in younger children. Therefore, effective prophylaxis requires frequent intravenous injection, usually three times per week or every other day. Several technologies are currently under investigation to extend the half-life of FVIII, including Fc fusion (Eloctate, Elocta, efmoroctocog alfa), addition of polyethylene glycol (turoctocog alfa pegol [N8-GP], BAY 94-9027, BAX 855), and a single-chain construct (CSL627). This review summarizes characteristics of products in clinical development and discusses their potential benefits.

The old and new: PCCs, VIIa, and long-lasting clotting factors for hemophilia and other bleeding disorders.

What is the correct use of established clotting factors, prothrombin complex concentrates (PCCs), and activated factor VII in bleeding complications of trauma, surgery, and old and new oral anticoagulants? How will new clotting factors, specifically the long-acting factors, change the hemostatic management of coagulation deficiency disorders? From bench to bedside, comparative coagulation studies and clinical trials of modified clotting factors are providing insights to help guide hemostatic management of congenital and acquired bleeding disorders. Comparative thrombin-generation studies and preclinical and clinical trials suggest that PCCs and fresh-frozen plasma are effective in reversing the anticoagulant effects of warfarin, yet there are few data to guide reversal of the new oral anticoagulants dabigatran and rivaroxaban. Although coagulation studies support the use of PCCs to reverse new oral anticoagulants, correlation with clinical response is variable and clinical trials in bleeding patients are needed. For congenital bleeding disorders, exciting new technologies are emerging from the bench. Data from clinical trials of molecularly modified coagulation factors with extended half-lives suggest the possibility of fewer infusions, reduced bleeds, and better quality of life in persons with hemophilia. Preclinical studies of other novel prohemostatic approaches for hemophilia and other congenital coagulation disorders include RNA interference silencing of antithrombin, monoclonal anti-tissue factor pathway inhibitor (anti-antibody, anti-tissue factor pathway inhibitor) aptamer, bispecific anti-IXa/X antibody, and fucoidans. Understanding the comparative coagulation studies of established prohemostatic agents, the pharmacokinetics of new long-acting clotting factors, and their correlation with bleeding outcomes will provide opportunities to optimize the hemostatic management of both congenital and acquired hemostatic disorders.

Low-dose menaquinone-7 supplementation improved extra-hepatic vitamin K status, but had no effect on thrombin generation in healthy subjects.

Vitamin K is required for the carboxylation of Gla-proteins in the liver (coagulation factors) and extra-hepatic tissues, such as bone (osteocalcin, OC), and arterial wall (matrix Gla-protein, MGP). Although the coagulation factors are essentially fully carboxylated under normal conditions, 10-40 % of OC and MGP remains undercarboxylated. We were therefore interested to study the dose-response effects of extra intake of menaquinones on the carboxylation of the extra-hepatic Gla-proteins. A total of forty-two healthy Dutch men and women aged between 18 and 45 years were randomised into seven groups to receive: placebo capsules or menaquinone-7 (MK-7) capsules at a daily dose of 10, 20, 45, 90, 180 or 360 µg. Circulating uncarboxylated OC (ucOC), carboxylated OC (cOC) and desphospho-uncarboxylated MGP were measured by ELISA. The ucOC:cOC ratio was calculated from circulating ucOC and cOC values. Endogenous thrombin potential and peak height were determined by calibrated automated thrombography. To increase the statistical power, we collapsed the treatment groups into three dosage groups: placebo, low-dose supplementation (doses below RDA, Commission Directive 2008/100/EC), and high-dose supplementation (doses around RDA, Commission Directive 2008/100/EC). MK-7 supplementation at doses in the order of the RDA (Commission Directive 2008/100/EC) increased the carboxylation of circulating OC and MGP. No adverse effects on thrombin generation were observed. Extra MK-7 intake at nutritional doses around the RDA (Commission Directive 2008/100/EC) improved the carboxylation of the extra-hepatic vitamin K-dependent proteins. Whether this improvement contributes to public health, i.e. increasing the protection against age-related diseases needs further investigation in specifically designed intervention trials.

Prohemostatic and antithrombin activities of Ankaferd hemostat are linked to fibrinogen gamma chain and prothrombin by functional proteomic analyses.

Ankaferd blood stopper (ABS) is a novel topical hemostatic agent of plant origin registered for the management of external hemorrhages, in Turkey. The ABS-induced formation of the protein network with vital erythroid aggregation covers the whole physiological hemostatic process. The aim of this study is to assess prohemostatic and antithrombin effects of ABS on the basis of functional proteomic analyses performed in ABS-treated plasma and serum samples based on the previous hypotheses about ABS action. For this purpose, serum and plasma proteins were separated by 2-dimensional (2D) gel electrophoresis, and proteins were identified using reference plasma gel on Swiss-2DPAGE database. Our results indicated that fibrinogen gamma chain and prothrombin levels just initially decreased first and thereafter enhanced following the ABS exposure. Dual effects of ABS on those critical hemostatic molecules seem to be associated with prohemostatic and antithrombin activities of the hemostatic agent.

Vitamin K supplementation increases vitamin K tissue levels but fails to counteract ectopic calcification in a mouse model for pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder in which calcification of connective tissue leads to pathology in skin, eye and blood vessels. PXE is caused by mutations in ABCC6. High expression of this transporter in the basolateral hepatocyte membrane suggests that it secretes an as-yet elusive factor into the circulation which prevents ectopic calcification. Utilizing our Abcc6 (-/-) mouse model for PXE, we tested the hypothesis that this factor is vitamin K (precursor) (Borst et al. 2008, Cell Cycle). For 3 months, Abcc6 (-/-) and wild-type mice were put on diets containing either the minimum dose of vitamin K required for normal blood coagulation or a dose that was 100 times higher. Vitamin K was supplied as menaquinone-7 (MK-7). Ectopic calcification was monitored in vivo by monthly micro-CT scans of the snout, as the PXE mouse model develops a characteristic connective tissue mineralization at the base of the whiskers. In addition, calcification of kidney arteries was measured by histology. Results show that supplemental MK-7 had no effect on ectopic calcification in Abcc6 ( -/- ) mice. MK-7 supplementation increased vitamin K levels (in skin, heart and brain) in wild-type and in Abcc6 (-/-) mice. Vitamin K tissue levels did not depend on Abcc6 genotype. In conclusion, dietary MK-7 supplementation increased vitamin K tissue levels in the PXE mouse model but failed to counteract ectopic calcification. Hence, we obtained no support for the hypothesis that Abcc6 transports vitamin K and that PXE can be cured by increasing tissue levels of vitamin K.

Effect of oxyethylene numbers on the pharmacokinetics of menatetrenone incorporated in oil-in-water lipid emulsions prepared with polyoxyethylene-polyoxypropylene block copolymers and soybean oil in rats.

We have prepared lipid emulsions of approximately 200 nm in diameter with soybean oil (SO) and a series of Pluronics with various numbers of oxyethylene units and about 60 oxypropylene units (SO/Pluronics), and studied the pharmacokinetics of menatetrenone incorporated into SO/Pluronics in rats. Emulsions of approximately 200 nm in diameter were obtained when SO contents were 2.5% and 20% (w/w) for 2.4% (w/w) PL101 and Pluronics that more than 30% was made up by oxyethylene units, respectively. The half-life of menatetrenone in plasma when oxyethylene units made up less than 30% of the Pluronic (SO/PL101 and SO/PP103) was similar to that for SO/egg yolk phosphatides (SO/EYP), but longer than that when oxyethylene units composed more than 40% of the Pluronic (SO/PP104 and SO/PF108, by 3- and 10-fold, respectively). Pretreatment with dextran sulfate 500000, an inhibitor of emulsion uptake by the reticuloendothelial system (RES), resulted in a higher plasma concentration and a lower liver uptake of menatetrenone as SO/PL101 at 10 min and SO/PP103 at 60 min, indicating that both SO/PL101 and SO/PP103 were taken up by the RES, although SO/PP103 required some time to be recognized by the RES. These findings suggested that larger numbers of oxyethylene units of Pluronics with 60 oxypropylene units were required for the longer plasma circulation of SO/Pluronics due to evasion of the RES.

Efficacy and safety of a new-class hemostatic drug candidate, AV513, in dogs with hemophilia A.

AV513 is a select fucoidan, a sulfated polysaccharide of botanical origin. It inhibits tissue factor pathway inhibitor (TFPI) activity and accelerates clotting of human hemophilia A and B plasma. In prior work, subcutaneous administration of AV513 to mice with hemophilia A improved hemostasis. The current studies were designed to evaluate potential efficacy and safety in dogs with hemophilia A (hemophilia A dogs) with minimally increased hemostasis after adenoassociated viral-FVIII gene transfer and in treatment-naive severe hemophilia A dogs. AV513 administered subcutaneously to low-FVIII dogs for multiple weeks improved hemostasis as exhibited in thromboelastography (TEG) and cuticle bleeding time (CBT) tests. Moreover, AV513 administered orally to AAV-FVIII dogs and treatment-naive severe hemophilia A dogs for a multiweek dose-escalating period yielded correction to normal ranges in both TEG and CBT end points at 5 to 15 mg/kg and 15 to 20 mg/kg dose levels, respectively. In all 3 separate studies, throughout their duration, AV513 was well tolerated by the dogs without any adverse events. Additional pharmacologic characterization of AV513 included intravenous pharmacokinetic analysis in rats. In summary, the combination of safety and efficacy in 2 global tests of hemostasis in the hemophilia A dog model indicate that further evaluation of AV513 as a hemostatic agent in hemophilia A patients is warranted.

Relative antithrombotic and antihemostatic effects of protein C activator versus low-molecular-weight heparin in primates.

The anticoagulant and anti-inflammatory enzyme, activated protein C (APC), naturally controls thrombosis without affecting hemostasis. We therefore evaluated whether the integrity of primary hemostasis was preserved during limited pharmacological antithrombotic protein C activator (PCA) treatment in baboons. The double-mutant thrombin (Trp215Ala/Glu217Ala) with less than 1% procoagulant activity was used as a relatively selective PCA and compared with systemic anticoagulation by APC and low-molecular-weight heparin (LMWH) at doses that inhibited fibrin deposition on thrombogenic segments of arteriovenous shunts. As expected, both systemic anticoagulants, APC (0.028 or 0.222 mg/kg for 70 minutes) and LMWH (0.325 to 2.6 mg/kg for 70 minutes), were antithrombotic and prolonged the template bleeding time. In contrast, PCA at doses (0.0021 to 0.0083 mg/kg for 70 minutes) that had antithrombotic effects comparable with LMWH did not demonstrably impair primary hemostasis. PCA bound to platelets and leukocytes, and accumulated in thrombi. APC infusion at higher circulating APC levels was less antithrombotic than PCA infusion at lower circulating APC levels. The observed dissociation of antithrombotic and antihemostatic effects during PCA infusion thus appeared to emulate the physiological regulation of intravascular blood coagulation (thrombosis) by the endogenous protein C system. Our data suggest that limited pharmacological protein C activation might exhibit considerable thrombosis specificity.

Oral absorption of ginsenoside Rb1 using in vitro and in vivo models.

This research attempts to clarify the cause for poor oral absorption of ginsenoside Rb1 (Rb1), one main ingredient of the well known Panax notoginseng saponins (PNS) for curing hemorrhage. Caco-2 cell monolayers were used as an in vitro model to reveal the transport mechanism of Rb1 across the intestinal mucosa. Moreover, the serum concentration-time profiles of Rb1 after tail venous (IV), portal venous (PV), intraduodenal (ID) and peroral (PO) administration to rats were compared to evaluate the first-pass effects of stomach, intestine and liver. In vitro experiments showed that uptake by Caco-2 cell monolayers was temperature dependent, but was not influenced by cyclosporine A and ketoconazole. The change in the apical pH showed no obvious effects on the uptake of Rb1. The uptake and transport were non-saturable, and flux from the apical compartment to the basolateral compartment (A-B) increased linearly with increasing concentration, which indicated a passive transport. Meanwhile, an apparent permeability coefficient of (5.90 +/- 1.02) x 10(-8) cm/s (C0 = 1 mg/mL) predicted an incomplete absorption. The investigation on the pharmacokinetic behavior of Rb1 after different routes of administration to rats showed a significant difference between PO (F(PO) was 0.64%), ID (F(ID) was 2.46%) and PV (F(PV) was 59.49%) administration, and the first-pass effect of the intestine is more significant than that of the stomach and liver in the absorption process. In summary, elimination in the stomach, large intestine and liver contributed to the poor absorption of Rb1, but the low membrane permeability might be a more important factor dominating the extent of absorption.

Effect of acyl chains of phosphatidylcholines on the pharmacokinetics of menatetrenone incorporated in O/W lipid emulsions prepared with phosphatidylcholines and soybean oil in rats.

Oil-in-water (O/W) lipid emulsions were prepared with phosphatidylcholines (PCs) of various acyl chains and soybean oil (SO) using a microfluidizer system, and the pharmacokinetics of menatetrenone incorporated in these oil particles were examined at the clinical injection volume (0.1 mL kg(-1)) in rats. The plasma half-life of menatetrenone incorporated in the oil particles prepared with SO and dipalmitoylphosphatidylcholine (DPPC) (SO/DPPC) was longer than that prepared with SO and eggyolk phosphatides (EYP) (SO/EYP) by 3 fold, while those of menatetrenone as oil particles prepared with SO and either dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC) or dilinoleoyl phosphatidylcholine (DLoPC) (SO/DLPC, SO/DMPC, SO/DSPC, SO/DOPC and SO/DLoPC, respectively) were similar to that of menatetrenone as SO/EYP. The menatetrenone uptake by the liver was not significantly different from that as SO/EYP in all SO/PCs examined, but the menatetrenone uptake by the spleen as SO/DPPC and SO/DSPC was higher than that as SO/EYP. The menatetrenone uptake by the lungs as SO/DPPC was also higher than that as SO/EYP. These findings suggest that SO/DPPC is a good candidate drug carrier for the prolonged plasma circulation of lipophilic drugs.

Prolonged circulation of menatetrenone by emulsions with hydrogenated castor oils in rats.

Previously, we reported that plasma half-lives of a drug incorporated in lipid emulsions prepared with soybean oil (SO), a long-chain triglyceride, and hydrogenated castor oils (HCOs) (SO/HCOs) were markedly longer, while those as SO/polyoxyethylene sorbitan esters (SO/PSs) were similar, compared to that as SO/egg yolk phosphatides (SO/EYP) [J. Pharm. Pharmacol. 54 (2002) 1357; J. Drug Target. 11 (2003) 37]. In the present study, lipid emulsions were prepared with Miglyol 812 (MO), a medium-chain triglyceride, and HCOs, and the kinetics of the incorporated drug, menatetrenone, were examined. The plasma half-lives and the liver uptake of menatetrenone as MO/polyoxyethylene-(10)-hydrogenated castor oils (MO/HCO10s) were similar to and larger than those as MO/EYP, respectively. On the other hand, the plasma half-lives and liver uptake of menatetrenone as MO/polyoxyethylene-(20)-hydrogenated castor oils (MO/HCO20s) or MO/polyoxyethylene-(60)-hydrogenated castor oils (MO/HCO60s) were markedly longer and lower than those as MO/EYP, respectively. The pretreatment of dextran sulfate 500,000, a reticuloendothelial system suppressor, raised the plasma concentration and inhibited liver uptake of menatetrenone as MO/HCO10, but not for MO/HCO20. These findings suggest that the minimum number of oxyethylene units within HCOs for the prolonged plasma circulation of menatetrenone was 20 for MO/HCOs, similarly to SO/HCOs.

Effect of oxyethylene moiety in polyoxyethylene sorbitan esters on the pharmacokinetics of menatetrenone incorporated in O/W lipid emulsions prepared with polyoxyethylene sorbitan esters and soybean oil in rats.

Oil-in-water (O/W) lipid emulsions are suitable drug carriers for lipophilic drugs; however, the effects of numbers or chains of oxyethylene units within a surfactant molecule such as polyoxyethylene sorbitan esters (PSs) on the biological fate of these lipid emulsions have not yet been clarified. In this study, a series of PSs and soybean oil (SO) were utilized to prepare menatetrenone-incorporated lipid emulsions (SO/PSs), and the biological fate of menatetrenone administered as SO/PSs was studied at a clinical injection volume (0.1 mL kg(-1)) in rats. The plasma concentration and organ uptake of menatetrenone administered as SO/20OE-PSs (PSs with 20 oxyethylene units) was similar to that of SO/egg-yolk phosphatides (SO/EYP). The plasma concentration of menatetrenone was extensively lower for SO/6OE-PSs (PSs with 6 oxyethylene units) and SO/20OE-3FA-PSs (PSs with 20 oxyethylene units and 3 fatty acid chains) than that for SO/EYP, and menatetrenone uptake by the liver and spleen was higher for SO/6OE-PSs and SO/20OE-3FA-PSs, respectively, than those for SO/EYP. Furthermore, menatetrenone uptake by the lungs was also increased for SO/6OE-PS and SO/20OE-3FA-PS with double bonds in the fatty acid moieties of the PSs. These findings suggested that shortening the oxyethylene units or decreasing the oxyethylene chain numbers of emulsifiers resulted in a rapid clearance of the lipid emulsions from the circulation by extensive uptake via the liver, spleen or lungs.

O/W lipid emulsions for parenteral drug delivery. III. Lipophilicity necessary for incorporation in oil particles even after intravenous injection.

The potential usefulness of oil-in-water (O/W) lipid emulsions as injectable drug delivery systems was examined. Plasma concentrations of oil particles after intravenous injection of a standard lipid emulsion composed of soybean oil and egg yolk phosphatides were monitored based on the plasma concentrations of phospholipids and triglycerides, and the light scattering intensity of the plasma. Their time profiles were similar to each other, and the oil particle size decreased time-dependently. Pretreatment with dextran sulfate, a known reticuloendothelial system (RES) suppressor, resulted in marked reduction of the plasma clearance of the oil particles and of the time-dependent alteration of oil particle size, suggesting that oil particles were trapped by RES. The lipophilicity of the drug needed for its incorporation in the oil particles even after intravenous injection was found to be clog P > 8, where clog P is the calculated logarithm of the partition coefficient between n-octanol and water. In the case of sudan II (clog P = 5.4), the release from the oil particles was very quick after intravenous injection, resulting in slight alteration in biodistribution when compared with its micellar solution. In contrast, menatetrenone (clog P = 9.5) was selectively delivered to the liver, lungs and spleen, being consistent with the oil particles taken up by RES.

A pharmacokinetic study with the high-dose anticancer agent menadione in rabbits.

The aim of this investigation was to assess the pharmacokinetic properties of high-dose menadione (VK3), as an anticancer agent, in plasma and red blood cells (RBCs) in rabbits. An extremely high dose of 75 mg menadiol sodium diphosphate (Synkayvite) was intravenously injected. HPLC analysis was applied to measure the major metabolite, menadione, VK3. The kinetic properties of VK3 in both plasma and red blood cells showed a short elimination half-life, high clearance, and large volume of distribution in plasma and RBCs. The mean elimination t1/2 values of menadione in plasma and in RBCs were 27.17 +/- 10.49 min and 35.22 +/- 11.82 min, respectively. The plasma clearance (CL/F) of VK3 was 0.822 +/- 0.254 L min-1. The systemic clearance in RBCs was 0.407 +/- 0.152 L min-1. The apparent volume of distribution (Vd/F) in plasma was 30.833 +/- 12.835 L and that in RBCs 20.488 +/- 9.401 L. The plasma AUC was 32.453 +/- 9.785 micrograms min mL-1 and that of RBCs 67.219 +/- 24.449 micrograms min mL-1. Menadiol was rapidly biotransformed to menadione in blood. The formation rate constant (kf) of menadione in plasma was 0.589 +/- 0.246 min-1, and that of RBCs 1.520 +/- 1.345 min-1. Through this study the estimated menadione dosage needed to maintain a plasma level of 1 microgram mL-1 for anticancer purposes was 19.7 mg kg-1 every hour.

[Determination of pharmacokinetic parameters of shandahuang xiaoyan zhixue capsules].

Based on residual accumulation and biophase availability principles and using animal death rate as index, determination has been carried out of the residual accumulation rate as well as the toxicokinetic and pharmacokinetic parameters at different times of the Shandahuang Xiaoyan Zhixue Capsules.