Conjugation of oxaliplatin with PEGylated-nanobody for enhancing tumor targeting and prolonging circulation.

Oxaliplatin is a platinum-based drug used in clinic for cancer chemotherapy. Despite of its success, the non-selective effect on normal cells causes severe side-effects and hampers its applications. Targeted delivery of oxaliplatin to cancer cells is an effective approach to enhance drug efficacy and reduce adverse effect. In this work, the Pt(IV) prodrug of oxaliplatin has been conjugated to poly(ethylene glycol) (PEG) modified nanobody in order to achieve tumor targeting as well as improved circulation in vivo. The Pt(IV) prodrug was site-specifically linked to an anti-epidermal growth factor receptor (EGFR) nanobody, so that the drug can be accumulated more pronounced in EGFR positive tumor cells than in normal cells. The effect of different length of PEG on the drug circulation has been investigated, while the fusion of anti-albumin nanobody was used for comparison. The result demonstrates that the prolonged drug circulation significantly increases the in vivo drug efficiency of the oxaliplatin-nanobody conjugate.

Surface loading of nanoparticles on engineered or natural erythrocytes for prolonged circulation time: strategies and applications.

Nano drug-delivery systems (DDS) may significantly improve efficiency and reduce toxicity of loaded drugs, but a few nano-DDS are highly successful in clinical use. Unprotected nanoparticles in blood flow are often quickly cleared, which could limit their circulation time and drug delivery efficiency. Elongating their blood circulation time may improve their delivery efficiency or grant them new therapeutic possibilities. Erythrocytes are abundant endogenous cells in blood and are continuously renewed, with a long life span of 100-120 days. Hence, loading nanoparticles on the surface of erythrocytes to protect the nanoparticles could be highly effective for enhancing their in vivo circulation time. One of the key questions here is how to properly attach nanoparticles on erythrocytes for different purposes and different types of nanoparticles to achieve ideal results. In this review, we describe various methods to attach nanoparticles and drugs to the erythrocyte surface, and discuss the key factors that influence the stability and circulation properties of the erythrocytes-based delivery system in vivo. These data show that using erythrocytes as a host for nanoparticles possesses great potential for further development.

The effect of zwitterionic surface content on blood circulation time of nanocapsule.

Zwitterionic modification can prolong the blood circulation time of nanocarrier in vivo, but zwitterionic content will affect the functions of nanocarrier such as enzyme-responsive and intracellular or extracellular delivery. Therefore, it is necessary to explore the relationship between the zwitterionic content and circulation time of nanocarrier so as to figure out what content of zwitterion can enable the nanocarrier to obtain both the long blood circulation ability and other functions mentioned above. Herein, using nanocapsule as a research model, we investigated the nanocapsule modified with zwitterion of phosphorylcholine (PC) or carboxybetaine (CB) respectively, and through 1H-NMR quantification we determined the zwitterionic surface content, so as to study the effect of PC or CB surface content on blood circulation performance of nanocapsule. In vivo study showed that the nanocapsule possessed an optimal surface filling ratios range for blood circulation of 43-68% for PC and of 20-68% for CB, with the longest t1/2=37.35 h for PC-nanocapsule and t1/2=45.27 h for CB-nanocapsule. Furthermore, the protein adsorption and macrophage endocytosis experiments indicated that when the surface filling ratio reached 43% for PC-nanocapsule and 20% for CB-nanocapsule, it could effectively reduce the protein adsorption and weaken macrophage endocytosis, thus explaining the phenomenon of long circulation time of nanocapsules from the point of protein adsorption and interaction with immune cells. This study proposes a new direction for designing long-circulating nanocarrier, and provides basis for constructing enzyme-responsive and intracellular or extracellular delivery platform.

Safety, tolerability, and efficacy of idarucizumab for the reversal of the anticoagulant effect of dabigatran in healthy male volunteers: a randomised, placebo-controlled, double-blind phase 1 trial.

BACKGROUND: Idarucizumab is a monoclonal antibody fragment that binds dabigatran with high affinity in a 1:1 molar ratio. We investigated the safety, tolerability, and efficacy of increasing doses of idarucizumab for the reversal of anticoagulant effects of dabigatran in a two-part phase 1 study (rising-dose assessment and dose-finding, proof-of-concept investigation). Here we present the results of the proof-of-concept part of the study. METHODS: In this randomised, placebo-controlled, double-blind, proof-of-concept phase 1 study, we enrolled healthy volunteers (aged 18-45 years) with a body-mass index of 18·5-29·9 kg/m(2) into one of four dose groups at SGS Life Sciences Clinical Research Services, Belgium. Participants were randomly assigned within groups in a 3:1 ratio to idarucizumab or placebo using a pseudorandom number generator and a supplied seed number. Participants and care providers were masked to treatment assignment. All participants received oral dabigatran etexilate 220 mg twice daily for 3 days and a final dose on day 4. Idarucizumab (1 g, 2 g, or 4 g 5-min infusion, or 5 g plus 2·5 g in two 5-min infusions given 1 h apart) was administered about 2 h after the final dabigatran etexilate dose. The primary endpoint was incidence of drug-related adverse events, analysed in all randomly assigned participants who received at least one dose of dabigatran etexilate. Reversal of diluted thrombin time (dTT), ecarin clotting time (ECT), activated partial thromboplastin time (aPTT), and thrombin time (TT) were secondary endpoints assessed by measuring the area under the effect curve from 2 h to 12 h (AUEC2-12) after dabigatran etexilate ingestion on days 3 and 4. This trial is registered with ClinicalTrials.gov, number NCT01688830. FINDINGS: Between Feb 23, and Nov 29, 2013, 47 men completed this part of the study. 12 were enrolled into each of the 1 g, 2 g, or 5 g plus 2·5 g idarucizumab groups (nine to idarucizumab and three to placebo in each group), and 11 were enrolled into the 4 g idarucizumab group (eight to idarucizumab and three to placebo). Drug-related adverse events were all of mild intensity and reported in seven participants: one in the 1 g idarucizumab group (infusion site erythema and hot flushes), one in the 5 g plus 2·5 g idarucizumab group (epistaxis); one receiving placebo (infusion site haematoma), and four during dabigatran etexilate pretreatment (three haematuria and one epistaxis). Idarucizumab immediately and completely reversed dabigatran-induced anticoagulation in a dose-dependent manner; the mean ratio of day 4 AUEC2-12 to day 3 AUEC2-12 for dTT was 1·01 with placebo, 0·26 with 1 g idarucizumab (74% reduction), 0·06 with 2 g idarucizumab (94% reduction), 0·02 with 4 g idarucizumab (98% reduction), and 0·01 with 5 g plus 2·5 g idarucizumab (99% reduction). No serious or severe adverse events were reported, no adverse event led to discontinuation of treatment, and no clinically relevant difference in incidence of adverse events was noted between treatment groups. INTERPRETATION: These phase 1 results show that idarucizumab was associated with immediate, complete, and sustained reversal of dabigatran-induced anticoagulation in healthy men, and was well tolerated with no unexpected or clinically relevant safety concerns, supporting further testing. Further clinical studies are in progress. FUNDING: Boehringer Ingelheim Pharma GmbH & Co KG.

Effectiveness of intraarterial administration of fasudil hydrochloride for preventing symptomatic vasospasm after subarachnoid hemorrhage.

OBJECTIVE: We examined the effect of intraarterial administration of fasudil hydrochloride (IAFC), a Rho kinase inhibitor, for the prevention of symptomatic vasospasm after SAH by evaluating cerebral circulation. METHODS: We evaluated IAFC cases of 57 sides of 38 patients (12 men and 26 women, average age 60.2 years old) diagnosed with aneurysmal subarachnoid hemorrhage (SAH) from February 2012 to November 2012. All cases were treated by clipping or coil embolization within 48 h after onset. Indication for IAFC was the existence of a spastic change on follow-up digital subtraction angiography (DSA) compared with that of onset. RESULTS: Clipping was performed in 30 cases and coil embolization in 8 cases. IAFC was performed an average of 6.6 days after onset. Color gradient mapping demonstrated reduction of the circulation time after IAFC compared with before IAFC on 39 sides, no change on 15 sides, and extension on 3 sides. Average arterial circulation time before IAFC was 2.25 ± 0.57 s and after IAFC was 1.95 ± 0.55 s. IAFC significantly shortened average arterial circulation (P = 0.005). No case developed symptomatic vasospasm after IAFC. CONCLUSION: IAFC significantly reduced the cerebral circulation time after aneurysmal SAH and might be effective for the prevention of symptomatic vasospasm.

The bifunctional liposomes constructed by poly(2-ethyl-oxazoline)-cholesteryl methyl carbonate: an effectual approach to enhance liposomal circulation time, pH-sensitivity and endosomal escape.

PURPOSE: A novel bifunctional liposome with long-circulating and pH-sensitive properties was constructed using poly(2-ethyl-oxazoline)-cholesteryl methyl carbonate (PEtOz-CHMC) in this study. METHODS: PEtOz-CHMC was synthesized and characterized by TLC, IR and (1)H-NMR. The obtained PEtOz lipid was inserted into liposomes by the post-insertion method. Through a series of experiments, such as drug release, tumor cell uptake, cytotoxicity, calcium-induced aggregation, pharmacokinetic experiments, etc., the pH-sensitive and long-circulating properties of PEtOzylated liposomes was identified. RESULTS: PEtOz-CHMC modified liposomes (PEtOz-L) showed increased calcein release at low pH. Flow cytometric analysis results showed that the fusion and cellular uptake of PEtOz-L could be promoted significantly at pH 6.4 compared with those at pH 7.4. Confocal laser scanning microscope observations revealed that PEtOz-L could respond to low endosomal pH and directly released the fluorescent tracer into the cytoplasm. MTT assays in HeLa cells demonstrated that doxorubicin hydrochloride (DOX) loaded PEtOz-L exhibited stronger anti-tumor activity in a medium at pH 6.4 than in a medium pH 7.4. PEtOz-L remained stable when these liposomes were incubated in calcium chloride solution. The cumulative calcein release rate of PEtOz-L was significantly lower than that of CL when the liposomes were dialysed in PBS. The pharmacokinetic experiments of liposomes in rats showed that t 1/2 and AUC of PEtOz-L were 4.13 times and 4.71 times higher than those of CL. CONCLUSIONS: PEtOzylated liposomes exhibits excellent long-circulating and pH-sensitive properties. Our results suggest that PEtOz is a promising biomaterial for the modification of liposome in drug delivery.

Lysis onset time as diagnostic rotational thromboelastometry parameter for fast detection of hyperfibrinolysis.

BACKGROUND: Rotational thromboelastometry is increasingly used to detect hyperfibrinolysis, which is a predictor of unfavorable outcome in patients with coagulation disturbances. In an in vitro study, the authors investigated which thromboelastometric hemostatic parameters could be valuable for fast diagnosis of the severity of hyperfibrinolysis and confirmed their findings in a patient population with hyperfibrinolysis. METHODS: Thromboelastometry was performed after adding increasing concentrations of tissue plasminogen activator (0 to 400 ng/ml) to citrated blood samples of 15 healthy volunteers. Lysis parameters included the clotting time, maximum clot firmness, maximum lysis, and lysis onset time (LOT). The relation of tissue plasminogen activator with the LOT was further investigated in a patient population with out-of-hospital cardiac arrest and hyperfibrinolysis. RESULTS: The LOT showed a dose-dependent association with increasing tissue plasminogen activator concentrations. Late, intermediate, or fulminant hyperfibrinolysis was associated with an average LOT (mean ± SD) of 42.7 ± 13.8, 23.2 ± 8.2, and 17.5 ± 4.6 min in the in vitro study and estimated 42.2 ± 8.3, 29.1 ± 1.2, and 14.6 ± 7.7 min in patients, respectively. The authors found a moderately negative correlation between patient plasma tissue plasminogen activator levels and the LOT (r = -0.67; P = 0.01). CONCLUSION: This study shows that the LOT may be used for fast detection of severe hyperfibrinolysis, with a better resolution than the maximum lysis, and should be further evaluated for optimization of therapeutic strategies in patients with severe clot breakdown.

Effect of dexmedetomidine vs. acepromazine-methadone premedication on limb to lung circulation time in dogs.

The study compared limb-to-lung circulation times (CT) in dogs under general anaesthesia after premedication with dexmedetomidine (DEX) or acepromazine-methadone (ACE-M). Healthy male and female dogs (n=20) were randomly assigned to receive acepromazine 0.04mg/kg and methadone 0.2mg/kg intramuscularly (IM), or DEX 0.01mg/kg IM. Anesthesia was induced with propofol and maintained with isoflurane at similar concentration in both groups. Mechanical ventilation was started immediately (20breaths/min; inspiratory to expiratory ratio 1:2) and tidal volume was adjusted to achieve an end-tidal CO2 concentration (PE'CO2) of between 3.9 and 5.3kPa. Ten minutes later arterial blood gas was analyzed and baseline data recorded for 3 minutes. A single dose of sodium bicarbonate 0,5mEq/kg was administered intravenously over 10 s starting with inspiration. Limb-to-lung CT was defined as the time interval between the start of bicarbonate injection and the recording of the highest PE'CO2. Following bicarbonate administration, PE'CO2 increased, and then rapidly decreased to baseline in both groups. CT was shorter in the ACE-M group (20±2.3 vs. 27±5.1s). Bodyweight was higher in the ACE-M group (30.6±3.9 vs. 23.3±6.8kg). Mean arterial blood pressure was higher in the DEX group (92±9 vs. 73±7mmHg) but premedication with DEX significantly prolonged CT compared to premedication with ACE-M.

Investigating the effect of polymeric approaches on circulation time and physical properties of nanobubbles.

PURPOSE: A challenge in the field of nanobubbles, including lipobubbles and polymeric nanobubbles, is identification of formulation approaches to enhance circulation time or "bubble life" in the specific organ to allow for organ visualization. The aim of this study was to investigate the potential of two specific preparation approaches, polymeric surface modification to lipobubbles and a one-step approach for the preparation of ionotropically originated polymeric hydrogel nanobubbles for the production of biocompatible, biodegradable, and sufficiently echogenic ('flexible') bubbles, preferably within the nanometer range, that possess an enhanced in vivo lifetime compared to an unmodified lipobubble to allow visualization of the lymph node vasculature. METHODS: In the first approach, formed liposomes (basic and polymerically enhanced) were sequentially layered with appropriate cationic and anionic polyelectrolytes followed by transformation into polymer-coated nanobubbles. In addition, a one-step approach was employed for the fabrication of ionotropically originated polymeric hydrogel bubbles. RESULTS: Bubble lifetime was marginally enhanced by self-deposition of polyelectrolytes onto the normal lipobubble, however, not significantly (P = 0.0634). In general, formulations possessing a higher ratio of anionic:cationic coats and highly anionic overall surface charge (-20.62 mV to -17.54 mV) possessed an enhanced lifetime. The improvement in bubble lifetime was significant when a purely polymeric polyionic hydrogel bubble shell was instituted compared to a normal unmodified lipobubble (P = 0.004). There was enhanced persistence of these systems compared to lipobubbles, attributed to the highly flexible, interconnected hydrogel shell which minimized gas leakage. The prolonged contrast signal may also be attributed to a degree of polymeric deposition/endothelial attachment. CONCLUSIONS: This study identified the relevance of polymeric modifications to nanobubbles for an improved circulating lifetime, which would be essential for application of these systems in passive drug or gene targeting via the enhanced permeability and retention effect.

Accelerated blood clearance phenomenon upon repeated injection of PEG-modified PLA-nanoparticles.

PURPOSE: We recently developed prostaglandin E(1) (PGE(1))-encapsulated nanoparticles, prepared with a poly(lactide) homopolymer (PLA, Mw = 17,500) and monomethoxy poly(ethyleneglycol)-PLA block copolymer (PEG-PLA) (NP-L20). In this study, we tested whether the accelerated blood clearance (ABC) phenomenon is observed with NP-L20 and other PEG-modified PLA-nanoparticles in rats. METHODS: The plasma levels of PGE(1) and anti-PEG IgM antibody were determined by EIA and ELISA, respectively. RESULTS: Second injections of NP-L20 were cleared much more rapidly from the circulation than first injections, showing that the ABC phenomenon was induced. This ABC phenomenon, and the accompanying induction of anti-PEG IgM antibody production, was optimal at a time interval of 7 days between the first and second injections. Compared to NP-L20, NP-L33s that were prepared with PLA (Mw = 28,100) and have a smaller particle size induced production of anti-PEG IgM antibody to a lesser extent. NP-L20 but not NP-L33s gave rise to the ABC phenomenon with a time interval of 14 days. NP-L33s showed a better sustained-release profile of PGE(1) than NP-L20. CONCLUSIONS: This study revealed that the ABC phenomenon is induced by PEG-modified PLA-nanoparticles. We consider that NP-L33s may be useful clinically for the sustained-release and targeted delivery of PGE(1).

A flow sensitive alternating inversion recovery (FAIR)-MRI protocol to measure hemispheric cerebral blood flow in a mouse stroke model.

Blood flow imaging is an important tool in cerebrovascular research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides three-dimensional noninvasive quantitative methods of cerebral blood flow (CBF) imaging, but these MRI techniques have not yet been validated for mice. The authors compared CBF imaging using flow sensitive alternating inversion recovery (FAIR)-MRI and (14)C-Iodoantipyrine (IAP)-autoradiography in a mouse model of acute stroke. Twenty-nine male 129S6/SvEv mice were subjected to filamentous left middle cerebral artery occlusion (MCAo). CBF imaging was performed with (14)C-IAP autoradiography and FAIR-MRI using two different anesthesia protocols, namely intravenous infusion of etomidate or inhalation of isoflurane, which differentially affect perfusion. Using (14)C-IAP autoradiography, the average CBF in ml/(100 g*min) was 160+/-34 (isoflurane, n=5) vs. and 59+/-21 (etomidate, n=7) in the intact hemisphere and 43+/-12 (isoflurane, n=5) vs. 36+/-12 (etomidate, n=7) in the MCAo hemisphere. Using FAIR-MRI, the corresponding average CBFs were 208+/-56 (isoflurane, intact hemisphere, n=7), 84+/-9 (etomidate, intact hemisphere, n=7), 72+/-22 (isoflurane, MCAo hemisphere, n=7) and 48+/-13 (etomidate, MCAo hemisphere, n=7). Regression analysis showed a strong linear correlation between CBF measured with FAIR-MRI and (14)C-IAP autoradiography, and FAIR-MRI overestimated CBF compared to autoradiography. FAIR-MRI provides repetitive quantitative measurements of hemispheric CBF in a mouse model of stroke.

Phosphodiesterase 5 inhibitor, zaprinast, selectively increases cerebral blood flow in the ischemic penumbra in the rat brain.

BACKGROUND: Guanosine 3', 5'-cyclic monophosphate (cGMP) acts as a relaxant second messenger in the cerebral vessels. cGMP-specific phosphodiesterase type 5 (PDE5) inhibitor increases intracellular cGMP levels. This study investigated the effect of the PDE5 inhibitor on the ischemic brain. METHODS: Regional cerebral blood flow (rCBF), cGMP concentration, and infarction volume were measured in the rat middle cerebral artery occlusion model. Ten minutes after ischemia, the animals received an intravenous (i.v.) infusion of vehicle (phosphate-buffered saline), PDE5 inhibitor, zaprinast (10 mg/kg), or nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP, 100 microg/kg). rCBF was measured continuously by laser-Doppler flowmetry in the ischemic penumbra of the ischemic and contralateral sides under continuous blood pressure monitoring. cGMP concentrations were determined using the enzyme immunoassay and infarct volumes were estimated by 2,3,5-triphenyltetrazolium chloride staining. RESULTS: The administration of zaprinast significantly increased rCBF in the ischemic brain compared with the pre-drug control value despite the decreased mean blood pressure, whereas it did not affect rCBF in the contralateral side. The cGMP concentration was significantly higher in the ischemic cortex compared with the contralateral side. SNAP infusion increased the cGMP concentration in the bilateral cortices to a similar extent. The volume of cerebral infarction was significantly decreased by zaprinast administration. CONCLUSIONS: The PDE5 inhibitor zaprinast may selectively increase CBF in the ischemic brain via increased cGMP levels, thus providing a new strategy against acute cerebral infarction.

The effect of hemopure on coagulation in clinically relevant concentrations.

Hemopure is a new colloidal blood substitute that may influence coagulation. We designed this study to examine the influence of this product on in vitro coagulation of whole blood by using the thrombelastograph (TEG). Blood samples from 20 volunteers were obtained. Hemopure was added to blood samples to obtain 0.5, 1, and 2 g/dL mixtures of Hemopure in blood. Control consisted of an undiluted sample and, for comparison, two samples diluted with volumes of lactated Ringer's solution (LR) equivalent to the two higher Hemopure dilutions. TEG with Hemopure at a concentration of 2 g/dL showed significantly shorter reaction and clot formation k times and an increased alpha angle compared with control. LR dilution with equivalent volume to 2 g/dL Hemopure solution also resulted in significantly shorter reaction and k times, as well as an increased alpha angle. Coagulation in samples with Hemopure at concentrations of 0.5 and 1 mg/dL did not vary significantly from control. Maximum amplitude did not vary significantly from control in any samples. The effect of Hemopure on TEG measures of coagulation is not significantly different from that of LR at clinically relevant concentrations.

Effect of mannitol on regional cerebral blood flow in patients with intracerebral hemorrhage.

AIM: To evaluate the regional cerebral blood flow (rCBF) changes following IV mannitol bolus in patients with intracerebral hemorrhage (ICH). METHODS: In a hospital based randomized placebo controlled study, 21 CT proven ICH patients with Glasgow coma scale (GCS) score of 5 or more were subjected to clinical evaluation including GCS and Canadian Neurological stroke (CNS) scale. Cranial SPECT study was undertaken before and 60 min after 20% mannitol 100 ml IV in 20 min or sham infusion. The SPECT images were semi-quantitatively analyzed and asymmetry index of basal ganglia, frontal, parietal and occipital regions were calculated. RESULTS: There were 12 patients in mannitol and nine in control group who were evenly matched for age, mean arterial blood pressure, GCS score and size of hematoma. Only one patient died in mannitol group. Following mannitol, GCS score improved in six, worsened in two and remained unaltered in four patients. In the control group, GCS improved in seven, worsened in none and was unchanged in two patients. SPECT studies revealed reduction in asymmetry index in basal ganglia in four, frontal region in six, parietal in four and occipital region in five patients in mannitol group. In control group, asymmetry index was reduced in basal ganglia in one, frontal and parietal region in three each and occipital region in five patients. These differences between control and study group were not significant. CONCLUSION: Mannitol does not seem to significantly change the regional cerebral blood flow (rCBF) in ICH patients as evaluated by SPECT study.

Antithrombotic activity of a 2-kDa heparin fragment in an experimental model of carotid artery thrombosis in rats.

The antithrombotic activity of a 2-kDa heparin fragment was studied in a rat model of common carotid artery thrombosis that causes a completely occlusive thrombus with cessation of the blood flow within 10-15 min. The compound reduced thrombus formation in a dose-dependent manner, starting from an intravenous dose of 5 mg kg-1. A dose of 20 mg kg-1 completely prevented thrombus formation and apparently induced the almost complete lysis of the already formed occlusive thrombus. At none of the doses used did the compound cause increased bleeding or the formation of haematomas. The present results indicate that low molecular weight heparins, which have an established, highly beneficial effect in venous thromboembolism, are also highly effective in an animal model of arterial thrombosis.