Self-Propelled Gemini-like LMWH-Scaffold Nanodrugs for Overall Tumor Microenvironment Manipulation via Macrophage Reprogramming and Vessel Normalization.

Angiogenesis is the hallmark of melanoma that nurtures the tumor microenvironment (TME) for rapid tumor progression. Vessel normalization could benefit melanoma treatment through TME reconstruction, while its limited duration and extent are still the drag. Herein, two kinds of look-like nanodrugs, called Gemini-like nanodrugs (GLnano), were constructed separately with the same scaffold of antiangiogenic low molecular weight heparin (LMWH) and mixed upon administration in vivo. For one, doxorubicin (DOX) was encapsulated into LMWH-chrysin nanodrug (LCY) with DSPE-PEG-anisamide decoration (D-LCA nanodrugs) for active targeting and direct cell killing toward melanoma cells. For another, matrix metalloproteinases (MMPs)-sensitive peptide was conjugated to LMWH to encapsulate celecoxib (Cel) (C-Lpep nanodrugs), disassembling in TME by MMPs and releasing Cel for M2-to-M1 reprogramming of tumor-associated macrophages. Our results showed that GLnano could remarkably elongate the vessel normalization window up to 12 days with the highest pericyte coverage of nearly 75%, compared to only 4 days by LCY monotherapy. Furthermore, GLnano could spontaneously form the "treatment-delivery" loop to promote nanodrugs toward deep tumor regions, leading to a potent tumor inhibition, metastasis prevention, and overall TME improvements.

Anticoagulation Therapy in Patients with Chronic Kidney Disease.

Patients with chronic kidney disease (CKD) are at increased risk for both thrombotic events and bleeding. The early stages of CKD are mainly associated with prothrombotic tendency, whereas in its more advanced stages, beside the prothrombotic state, platelets can become dysfunctional due to uremic-related toxin exposure leading to an increased bleeding tendency. Patients with CKD usually require anticoagulation therapy for treatment or prevention of thromboembolic diseases. However, this benefit could easily be offset by the risk of anticoagulant-induced bleeding. Treatment of patients with CKD should be based on evidence from randomized clinical trials, but usually CKD patients are excluded from these trials. In the past, unfractionated heparins were the anticoagulant of choice for patients with CKD because of its independence of kidney elimination. However, currently low-molecular-weight heparins have largely replaced the use of unfractionated heparins owing to fewer incidences of heparin-induced thrombocytopenia and bleeding. We undertook this review in order to explain the practical considerations for the management of anticoagulation in these high risk population.

Effects of Chitosan Derivative N-[(2-Hydroxy-3-Trimethylammonium)Propyl]Chloride on Anticoagulant Activity of Guinea Pig Plasma.

Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 70 aIIa U/kg non-fractionated heparin shortened plasma clotting time (shown by partial activated thromboplastin time, thrombin time, and prothrombin time). Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 1 mg/kg (100 aXa U/kg) low-molecular-weight heparin (clexane) led to shortening of plasma clotting time in the ReaClot Heparin test and to prolongation of plasma amidolytic activity in the factor Xa chromogenic substrate test.

Thrombin generation assay identifies individual variability in responses to low molecular weight heparin in pregnancy: implications for anticoagulant monitoring.

Low molecular weight heparin (LMWH) given to inhibit coagulation and reduce the risk of thrombosis, is typically monitored by anti-Xa assay. However, anti-Xa levels may not necessarily provide an accurate measure of coagulation inhibition. Moreover, pregnancy is associated with hypercoagulability, which may compromise the efficacy of LMWH. We looked at the association between anti-Xa levels and parameters of thrombin generation assay [TGA; area under the curve (AUC), peak height (PH) and time to peak (ttP)] using samples from 41 pregnant women receiving LMWH and 40 normal pregnant women controls. TGA results confirmed the physiological hypercoagulability of normal pregnancy (mean normalised values: AUC 119%; PH 157%; ttP 72%). Although anti-Xa measures correlated with all three TGA parameters, this group correlation masked significant inter-individual variability, demonstrated by the R(2) value or coefficient of determination. Anti-Xa levels contributed to 74% of variation in AUC values, 63% of variation in PH values and only 53% of variation in ttP values. The remainder reflects the contribution of patients' intrinsic coagulation status. Hence, some patients with 'safe' anti-Xa levels may potentially be under-anticoagulated, particularly in pregnancy. Measuring coagulability directly with TGA may lower the risk of adverse events due to under-anticoagulation in selected patients.

A systematic review on the accumulation of prophylactic dosages of low-molecular-weight heparins (LMWHs) in patients with renal insufficiency.

PURPOSE: Although therapeutic dosages of most low-molecular-weight heparins (LMWHs) are known to accumulate in patients with renal insufficiency, for the lower prophylactic dosages this has not been clearly proven. Nevertheless, dose reduction is often recommended. We conducted a systematic review to investigate whether prophylactic dosages of LMWH accumulate in renal insufficient patients. METHODS: A comprehensive search was conducted on 17 February 2015 using Embase, Medline, Web of Science, Scopus, Cochrane, PubMed publisher, and Google scholar. The syntax emphasized for LMWHs, impaired renal function, and pharmacokinetics. The search yielded 674 publications. After exclusion by reading the titles, abstracts, and if necessary the full paper, 11 publications remained. RESULTS: For dalteparin and tinzaparin, no accumulation was observed. Enoxaparin, on the other hand, did lead to accumulation in patients with renal insufficiency, although not in patients undergoing renal replacement therapy. Bemiparin and certoparin also did show accumulation. No data were available for nadroparin. CONCLUSIONS: In this systematic review, we show that prophylactic dosages of tinzaparin and dalteparin are likely to be safe in patients with renal insufficiency and do not need dose reduction based on the absence of accumulation. However, prophylactic dosages of enoxaparin, bemiparin, and certoparin did show accumulation in patients with a creatinine clearance (CrCl) below 30 ml/min, and therefore, dose reduction is required. The differences in occurrence of accumulation seem to depend on the mean molecular weight of LMWHs.

Pharmacokinetics of Certoparin During In Vitro and In Vivo Dialysis.

The efficacy and safety of certoparin in the prophylaxis of clotting during hemodialysis have recently been proven. Different to other low-molecular weight heparins (LMWHs), certoparin does not accumulate in maintenance dialysis patients for unknown reasons. The purpose of the present study was to examine the impact of the dialysis procedure on the removal of certoparin. In a subgroup of the MEMBRANE study consisting of 12 patients, the pharmacokinetics of certoparin during hemodialysis was determined by means of the anti-Xa activity. In addition, the elimination of certoparin into continuously collected dialysate was assessed. Further, in vitro experiments with human blood-simulating high-flux hemodialysis and hemofiltration were performed to quantify the elimination and the sieving coefficients SK of the two LMWHs certoparin and enoxaparin compared with unfractionated heparin (UFH). The surrogate marker middle molecules inulin and myoglobin served as reference solutes during the experiments. Finally, the adsorption of (125) iodine-radiolabeled certoparin onto the synthetic dialysis membrane was quantified. The clinical study reconfirmed the absence of bioaccumulation of certoparin with anti-Xa activities between <0.01 and 0.02 IU/mL after 24 h. A short plasma half-life time of 2.0 ± 0.7 h was determined during hemodialysis. Of the total certoparin dose injected intravenously prior to hemodialysis, only 2.7% was eliminated into dialysate. The in vitro experiments further revealed only 6% of certoparin to be adsorbed onto the dialysis membrane. The anti-Xa activities of certoparin and enoxaparin slowly declined during in vitro hemofiltration to 87.3 ± 5.5 and 82.5 ± 9.4% of baseline, respectively, while inulin and myoglobin concentrations rapidly decreased. The anti-Xa activity of UFH remained unchanged. The SK of both LMWH and UFH was very low in hemofiltration and particularly in hemodialysis with values ≤0.1. The elimination kinetics during hemodialysis suggests strong protein-binding of certoparin. Different from LMWH significantly cleared by the kidneys, the relatively short half-life time of certoparin of only 2 h during hemodialysis allows a more reliable control of the anti-coagulatory effects and decreases the risk of bleeding complications. Dialytic removal does not significantly contribute to the clearance of certoparin in maintenance dialysis patients.

Preliminary safety evaluation of a taurocholate-conjugated low-molecular-weight heparin derivative (LHT7): a potent angiogenesis inhibitor.

In our previous studies, taurocholic acid (TA)-conjugated low-molecular-weight heparin derivative (LHT7) has been proven to be a potent anti-angiogenic agent by demonstrated successful blockage capability of vascular endothelial growth factors (VEGF). Preliminary safety evaluations were conducted based on its mechanism of action and chemical behavior. For this purpose, acute toxicity study, and hematological and serological evaluations were carried out. Additionally, in order to evaluate mechanism-related side effects, both blood pressure and the occurrence of proteinuria were measured using a treatment regime of multiple high doses of LHT7 in a biodistribution study. LD50 values for LHT7 in female and male mice were 56.9 and 64.7 mg kg(-1) doses, respectively. There were no vital fluctuations in the serological and hematological parameters, except for the elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 100 and 200 mg kg(-1) doses of LHT7, representing vital changes in the liver function. Moreover, the results of mechanism-related studies showed that blood pressure at 50 mg kg(-1) did not change but showed elevated levels of protein in urine. In the biodistribution study, a slight accumulation of LHT7 in the kidney and the liver were observed at the 50 mg kg(-1) repeated dose owing to the presence of bile acid. No fatal damage was observed in this study; most observations were related to the chemical composition or the mechanism of action of the material.

Low-molecular-weight heparin biosimilars: potential implications for clinical practice. Australian Low-Molecular-Weight Heparin Biosimilar Working Group (ALBW).

A working group of clinicians and scientists was formed to review the clinical considerations for use of low-molecular-weight heparin (LMWH) biosimilars. LMWH are biological molecules of significant complexity; the full complexity of chemical structure is still to be elucidated. LMWH biosimilars are products that are biologically similar to their reference product and rely on clinical data from a reference product to establish safety and efficacy. The complex nature of LMWH molecules means that it is uncertain whether a LMWH biosimilar is chemically identical to its reference product; this introduces the possibility of differences in activity and immunogenicity. The challenge for regulators and clinicians is to evaluate the level of evidence required to demonstrate that a LMWH is sufficiently similar to the reference product. The consensus opinion of the working group is that prior to clinical use a LMWH biosimilar should have proven efficacy and safety, similar to the reference product with prospective studies, which should be confirmed with a proactive post-marketing pharmacovigilance programme.

Tinzaparin, an alternative to subcutaneous unfractionated heparin, in patients with severe and end-stage renal impairment: a retrospective observational single-center study.

BACKGROUND: Tinzaparin could be easier to manage than unfractionated heparin in patients with severe renal impairment. However, clinical and pharmacologic data regarding its use in such patients are lacking. OBJECTIVES: The aims of this study were to determine, in patients with estimated glomerular filtration rate (eGFR) of <30 mL.min⁻1, tinzaparin pharmacokinetics (PK) parameters using a population PK approach and bleeding and thrombotic complications. METHODS: We performed a retrospective observational single-center study, including in-patients with eGFR of <30 mL.min⁻1 receiving prophylactic (4500 IU.d⁻1) or therapeutic (175 IU.kg⁻1.d⁻1) tinzaparin. Measured anti-Xa levels were analyzed using a nonlinear mixed-effects modeling approach. Individual predicted tinzaparin exposure markers at steady state were calculated for each patient and dosing regimen. The PK was also evaluated through Monte Carlo simulations based on the final covariate model parameter estimates. RESULTS: Over a 22-month period, 802 tinzaparin treatment periods in 623 patients were analyzed: two-thirds received a prophylactic dose, 66% had an eGFR of <20 mL.min⁻1, and 25% were on renal replacement therapy. In patients for whom anti-Xa measurements were performed (n = 199; 746 values), PK parameters, profiles, and maximum plasma concentrations were comparable with those in patients without renal impairment or in healthy volunteers. In the whole population, major bleeding occurred in 2.4% and 3.5% of patients receiving prophylactic and therapeutic doses over a median 9- and 7-day treatment period, respectively. No patients had thrombotic complications. CONCLUSION: Tinzaparin PK parameters and profiles were not affected by renal impairment. This suggests that tinzaparin, at therapeutic or prophylactic dose, could be an alternative to unfractionated heparin in hospitalized patients with severe renal impairment.

Market entry of biosimilar low-molecular-weight heparins in Europe: opportunities and challenges.

This article examines the market entry of biosimilar low-molecular-weight heparins (LMWHs) in Europe by focusing on regulatory requirements, pricing, reimbursement, prescribing, and dispensing. The window for biosimilar LMWHs to enter the market is narrow on the supply side because of several factors. These include (1) regulatory requirements, including a quality dossier, clinical and nonclinical studies, pharmacodynamic and pharmacokinetic studies, immunogenicity studies, and a comparability exercise (but a reduction in clinical data requirements might be plausible in some cases); (2) prices of originator LMWHs are lower than those of other biologic products; (3) European prices of originator LMWHs are lower than those observed in the rest of the world; (4) research and development and manufacturing costs are substantial; (5) costs of active pharmaceutical ingredients have increased following the heparin contamination crisis; and (6) biosimilar LMWHs may be subjected to generic medicine pricing regulations. Furthermore, there are limited opportunities for biosimilar LMWHs on the demand side. This is because, although LMWHs have a large market volume in Europe, demand-side incentives for biosimilar LMWHs are largely absent, and the questions about interchangeability and substitution between originator and biosimilar LMWHs have yet to be fully resolved.

Recent advances in search of oral heparin therapeutics.

Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are first choices of clinicians among available anticoagulants. Currently, these agents are administered either parenterally or subcutaneously, which reduces patient compliance and acceptability. Oral heparin may serve as an alternative to both parenteral heparin as well as presently available oral anticoagulants such as warfarin. This review focuses mainly upon recent perspectives in the development of heparin as an oral anticoagulant. The possibility of its success with special emphasis to nanotechnological approaches has been elaborated. Important strategies such as the use of penetration enhancers, the development of lipid conjugates of heparin, and the incorporation of heparin in polymeric matrix systems have been discussed. Additionally, introductory information on biological activities, physiochemical aspects, and pharmacokinetic and pharmacodynamic parameters of heparin is summarized. A brief comparison of UFH and LMWH is also included for reader's benefit. Informative discussion on clinical trials with the successes and limitations of oral heparin formulations is also presented. Overall, the present review provides complete insight to the research that has been carried out for the development of heparin as oral anticoagulant.

Tinzaparin versus dalteparin for periprocedure prophylaxis of thromboembolic events in hemodialysis patients: a randomized trial.

BACKGROUND: Low-molecular-weight heparin (LMWH) is cleared predominantly by the kidneys and hence there is uncertainty about the safety of its use in hemodialysis (HD) patients. Our primary objective was to compare whether tinzaparin and dalteparin differentially accumulate in HD patients. STUDY DESIGN: Open-label randomized controlled trial. SETTING & PARTICIPANTS: HD patients undergoing periprocedure bridging anticoagulation. INTERVENTION: After warfarin therapy was discontinued, participants were randomly assigned to either 3 daily doses of tinzaparin (175 IU/kg) or dalteparin (200 IU/kg), with 2 intervening HD treatments between the first dose of study drug and their procedure. OUTCOMES: The primary outcome was predialysis anti-Xa levels 20 to 24 hours after the third LMWH dose (therapeutic target, <0.2 IU/mL). Secondary outcomes included thromboembolic events and major bleeding. RESULTS: Of 29 eligible and consenting patients, 17 patients received tinzaparin and 12 patients received dalteparin. Mean predialysis anti-Xa level 20-24 hours after the third LMWH dose was 0.37 ± 0.23 (SD) IU/mL for tinzaparin and 0.62 ± 0.41 IU/mL for dalteparin (P = 0.1), indicating clinically important accumulation for both drugs. No invasive procedures were canceled due to study drug accumulation. 4 patients experienced serious adverse events (1 major bleed after traumatic arteriovenous fistula puncture in the tinzaparin arm, 2 non-ST-elevation myocardial infarctions [1 in each group], and 1 upper-extremity deep venous thrombosis [dalteparin group]). LIMITATIONS: Small sample size. CONCLUSIONS: Dalteparin and tinzaparin significantly accumulate in HD patients at therapeutic doses. "Bridging therapy" with LMWHs at therapeutic doses in HD patients who require temporary interruption of warfarin therapy has the potential for complications and is of uncertain benefit. Other anticoagulation strategies, including no bridging therapy or intravenous heparin, need comparative evaluation in this unique patient population.

[Safety and efficacy of various regimens of heparin-mediated prevention of venous thromboses in patients with intracranial haemorrhage].

Analysed in the article is the incidence rate of the development of venous thromboembolic complications in a total of 79 patients presenting with various-aetiology intracranial haemorrhage in different regimens of heparin-mediated prevention. The authors have revealed that early (on day 2-4 after the onset of the disease) administration of preventive doses of heparin in patients with intracerebral and intracranial haematomas is a safe and efficient regimen, since it decreases the rate of venous thromboses in the system of the vena cava inferior and fatal thromboembolic complications as compared with a later term (on day 5 and more) of initiating heparin-mediated prevention.

Biomimetic solid lipid nanoparticles for oral bioavailability enhancement of low molecular weight heparin and its lipid conjugates: in vitro and in vivo evaluation.

Low molecular weight heparin (LMWH) is an anionic oligosaccharide macromolecule, which is commonly administered via parenteral routes for the treatment of vascular disorders like deep vein thrombosis (DVT) and pulmonary embolism (PE). Oral heparin delivery can tremendously improve the treatment of such disorders but is restricted due to its large size and anionic character. The present investigation describes synthesis of LMWH-lipid conjugates and their encapsulation in phosphatidylcholine stabilized biomimetic solid lipid nanoparticles (SLNs) for LMWH's oral bioavailability enhancement. Briefly, LMWH was conjugated with different saturated lipids of varying chain length (stearic acid, palmitic acid and myristic acid) using carbodiimide chemistry. The conjugation was confirmed with IR and (1)H NMR spectroscopy. The LMWH-lipid conjugate loaded SLNs were characterized for various parameters like shape, size, zeta potential, entrapment efficiency and in vitro release behavior in different simulated GIT pH mediums. The GIT toxicity of LMWH-lipid conjugate loaded SLNs to different tissues of intestinal epithelium was observed using H&E staining followed by microscopic observation at cellular level. The LMWH-lipid conjugate loaded SLNs were found to be safe for oral administration. The plasma concentration of LMWH was estimated using anti-FXa chromogenic assay. A significantly higher bioavailability (p < 0.05) of LMWH was observed using LMWH-lipid conjugates loaded SLNs in comparison to LMWH loaded SLNs and free LMWH. The order of different conjugates in bioavailability enhancement was LMWH-stearic acid > LMWH-palmitic acid > LMWH-myristic acid. This strategy holds promise for future applications of oral delivery of LMWH conjugates in the form of SLNs particularly for the treatment of cardiovascular disease like DVT and PE.

The therapeutic equivalence of complex drugs.

When the patent of a small molecule drug expires generics may be introduced. They are considered therapeutically equivalent once pharmaceutical equivalence (i.e. identical active substances) and bioequivalence (i.e. comparable pharmacokinetics) have been established in a cross-over volunteer study. However this generic paradigm cannot be applied to complex drugs as biologics and a number of other therapeutic modalities. For copies of biologics the European Medicine Agency and other regulatory agencies have introduced a new regulatory biosimilar pathway which mandates clinical trials to show therapeutic equivalence. However for other complex drugs such as the iron-carbohydrate drugs, low molecular weight heparins (LMWHs), liposomal drugs and the glatiramoids regulatory guidance is still mostly lacking. In this paper we will discuss (therapeutic) experience obtained so far with these different classes of 'complex drugs' and their specifics to provide scientific arguments and criteria for consideration for a regulatory framework for the market authorization for these type of drugs.

Transdermal delivery of low molecular weight heparin loaded in flexible liposomes with bioavailability enhancement: comparison with ethosomes.

Low molecular weight heparin (LMWH)-loaded flexible liposomes (flexosomes) were formulated for transdermal delivery, and their physicochemical and pharmacokinetic parameters were compared with LMWH-loaded ethosomes. Flexosomes had similar particle size compared with ethosomes, but their deformability was higher than that of ethosomes (76.7% vs. 46.8%). In vitro, flexosomes demonstrated 2.6-fold higher permeability coefficient than ethosomes. In comparison to LMWH aqueous solution, skin deposition of flexosome increased 3.2-fold, while that of ethosome increased only 2.0-fold. In vivo, after the topical application of flexosome to hairless mouse, [anti-Xa](max) was 1.11 IU/mL, while ethosomes showed only 0.32 IU/mL. Moreover, AUC(0-24 h) of flexosomes was 2.5-fold higher than ethosomes. In conclusion, the enhanced skin permeation and bioavailability of LMWH can be achieved with flexosomes in comparison with ethosomes. The LMWH transdermal delivery via flexosomes has the potential to replace the parenteral dosage forms for the treatment of venous thromboembolism, pulmonary embolism and cardiovascular events.

Thrombosis prophylaxis in critically ill patients.

Incidence of deep vein thrombosis in critically ill patients depends on the underlying disease but may be as high as 60%. The Surviving Sepsis Campaign clearly recommends administering anticoagulation in the absence of specific contraindications in patients with severe sepsis or septic shock. The article discusses risk factor for thromboembolic events in critical illness as well as means of non-pharmacologic and pharmacologic thrombosis prophylaxis. Peripheral vasoconstriction, edema, shock, and administration of catecholamines may reduce the bioavailability and efficacy of subcutaneous administration of low molecular weight heparin. This article further elaborates on the problem and pathophysiology of heparin resistance. Continuous intravenous administration of new anticoagulants may be a promising alternative to indirect anticoagulants. Severity of illness and SAPS II-score determine dosing of the direct thrombin inhibitor argatroban which needs to be about 10-times lower than in patients without critical illness.