An Investigation of the Assay of Heparin Based on Its Activity in Vivo

1967 ◽  
Vol 18 (01/02) ◽  
pp. 150-160 ◽  
Author(s):  
W. E Rezansoff ◽  
L. B Jaques
Keyword(s):  
Lipolytic Activity ◽  
Area Under The Curve ◽  
Test System ◽  
Assay Method ◽  
In Vitro Tests ◽  
Clotting Time ◽  
Oil Emulsion ◽  
Effective Doses

SummaryThe potency of different heparin preparations was investigated in vivo, by measuring the Lee and White clotting time, lipolytic activity and partial thromboplastin time of blood samples drawn at intervals after the intravenous injection of heparin into the anesthetized dog. The blood clotting time in log minutes, the thromboplastin time in log seconds, and the plasma lipolytic activity measured as the decrease in optical density of a synthetic cocuont oil emulsion per unit of time after incubation with postheparin plasma, were plotted against time after injection. A linear relation was obtained between the dose of heparin and the response measured as the area under the curve.The in vivo responses for 6 heparin preparations at 3 dose levels were compared with those for a reference heparin. The potency of each relative to the reference was estimated as the ratio of equally effective doses. The results obtained were compared with assay values reported for in vitro tests - U.S.P. , Howell, colorimetric (Lovibond, Beckman DK-2 spectrophotometer and microelectrophoresis on agarose) . There was no consistent case of one assay methodgiving results greatly different from the others and no assay method consistently described the activity of the heparin preparation in another test system, in vitro or in vivo.

scholarly journals Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action

2020 ◽  
Author(s):  
Katherine E. Chapman ◽  
Eleanor C. Wilde ◽  
Fiona M. Chapman ◽  
Jatin R. Verma ◽  
Ume-Kulsoom Shah ◽  
...  
Keyword(s):  
Cell Line ◽  
Human Cell Line ◽  
Test System ◽  
In Vitro Tests ◽  
Correct Identification ◽  
In Vitro Test ◽  
Human Lymphoblastoid Cell ◽  
Carcinogenic Potential

Abstract Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound’s subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001–770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the “misleading” in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.

scholarly journals Lymphatic absorption of α-linolenic acid in rats fed flaxseed oil-based emulsion

2010 ◽  
Vol 105 (7) ◽  
pp. 1026-1035 ◽  
Author(s):  
Leslie Couëdelo ◽  
Carole Boué-Vaysse ◽  
Laurence Fonseca ◽  
Emeline Montesinos ◽  
Sandrine Djoukitch ◽  
...  
Keyword(s):  
Linolenic Acid ◽  
Area Under The Curve ◽  
Flaxseed Oil ◽  
Lymphatic Absorption ◽  
Oil Emulsion ◽  
Emulsion Group ◽  
Lymph Duct ◽  
Acidic Conditions

The bioavailability of α-linolenic acid (ALA) from flaxseed oil in an emulsified formv.a non-emulsified form was investigated by using two complementary approaches: the first one dealt with the characterisation of the flaxseed oil emulsion inin vitrogastrointestinal-like conditions; the second one compared the intestinal absorption of ALA in rats fed the two forms of the oil. Thein vitrostudy on emulsified flaxseed oil showed that decreasing the pH from 7·3 to 1·5 at the physiological temperature (37°C) induced instantaneous oil globule coalescence. Some phase separation was observed under acidic conditions that vanished after further neutralisation. The lecithin used to stabilise the emulsions inhibited TAG hydrolysis by pancreatic lipase. In contrast, lipid solubilisation by bile salts (after lipase and phospholipase hydrolysis) was favoured by preliminary oil emulsification. Thein vivoabsorption of ALA in thoracic lymph duct-cannulated rats fed flaxseed oil, emulsified or non-emulsified, was quantified. Oil emulsification significantly favoured the rate and extent of ALA recovery as measured by the maximum ALA concentration in the lymph (Cmax = 14 mg/ml at 3 h in the emulsion groupv.9 mg/ml at 5 h in the oil group;P < 0·05). Likewise, the area under the curve of the kinetics was significantly higher in the emulsion group (48 mg × h/ml for rats fed emulsionv.26 mg × h/ml for rats fed oil;P < 0·05). On the whole, ALA bioavailability was improved with flaxseed oil ingested in an emulsified state. Data obtained from thein vitrostudies helped to partly interpret the physiological results.

scholarly journals Comparative Studies on the Anticoagulant Profile of Branded Enoxaparin and a New Biosimilar Version

2020 ◽  
Vol 26 ◽  
pp. 107602962096082
Author(s):  
Dalia Qneibi ◽  
Eduardo Ramacciotti ◽  
Ariane Scarlatelli Macedo ◽  
Roberto Augusto Caffaro ◽  
Leandro Barile Agati ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Thrombin Generation ◽  
Area Under The Curve ◽  
Factor Xa ◽  
In Vivo Studies ◽  
In Vitro Tests ◽  
Thrombin Time

Low molecular weight heparins (LMWH) represent depolymerized heparin prepared by various methods that exhibit differential, biochemical and pharmacological profiles. Enoxaparin is prepared by benzylation followed by alkaline depolymerization of porcine heparin. Upon the expiration of its patent, several biosimilar versions of enoxaparin have become available. Heparinox (Sodic enoxaparine; Cristália Produtos Químicos Farmacêuticos LTDA, Sao Paulo, Brazil) is a new biosimilar form of enoxaparin. We assessed the molecular weight and the biochemical profile of Heparinox and compared its properties to the original branded enoxaparin (Lovenox; Sanofi, Paris, France). Clotting profiles compared included activated clotting time, activated partial thromboplastin time (aPTT), and thrombin time (TT). Anti-protease assays included anti-factor Xa and anti-factor IIa activities. Thrombin generation was measured using a calibrated automated thrombogram and thrombokinetic profile included peak thrombin, lag time and area under the curve. USP potency was determined using commercially available assay kits. Molecular weight profiling was determined using high performance liquid chromatography. We determined that Heparinox and Lovenox were comparable in their molecular weight profile. Th anticoagulant profile of the branded and biosimilar version were also similar in the clot based aPTT and TT. Similarly, the anti-Xa and anti-IIa activities were comparable in the products. No differences were noted in the thrombin generation inhibitory profile of the branded and biosimilar versions of enoxaparin. Our studies suggest that Heparinox is bioequivalent to the original branded enoxaparin based upon in vitro tests however will require further in vivo studies in animal models and humans to determine their clinical bioequivalence.

scholarly journals Predicting and Testing Bioavailability of Magnesium Supplements

Nutrients ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 1663 ◽  
Author(s):  
Laura Blancquaert ◽  
Chris Vervaet ◽  
Wim Derave
Keyword(s):  
Serum Magnesium ◽  
Area Under The Curve ◽  
In Vitro Tests ◽  
In Vitro Test ◽  
Beneficial Effects ◽  
Dissolution Tests ◽  
Acute Ingestion ◽  
In Vivo Testing

Despite the presumption of the beneficial effects of magnesium supplementation, little is known about the pharmacokinetics of different magnesium formulations. We aimed to investigate the value of two in vitro approaches to predict bioavailability of magnesium and to validate this in subsequent in vivo testing. In vitro assessment of 15 commercially available magnesium formulations was performed by means of a Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and by dissolution tests. Two magnesium formulations with contrasting bioavailability prediction from both in vitro tests (best vs. worst) were selected for in vivo testing in 30 subjects. In vivo bioavailability was compared following one acute ingestion by monitoring blood magnesium concentrations up to 6 h following intake. The in vitro tests showed a very wide variation in absorption and dissolution of the 15 magnesium products. In the in vivo testing, a significant different serum magnesium absorption profile was found up to 4 h following supplement ingestion for the two supplements with opposing in vitro test results. Moreover, maximal serum magnesium increase and total area under the curve were significantly different for both supplements (+6.2% vs. +4.6% and 6.87 vs. 0.31 mM.min, respectively). Collectively, poor bioaccessibility and bioavailability in the SHIME model clearly translated into poor dissolution and poor bioavailability in vivo. This provides a valid methodology for the prediction of in vivo bioavailability and effectiveness of micronutrients by specific in vitro approaches.

Lupus-Like Anticoagulant In A Patient With Severe Factor VIII Deficiency

1981 ◽  
Author(s):  
R S Weinger ◽  
J L Moake ◽  
D Deykin ◽  
A Lopez ◽  
J D Olson
Keyword(s):  
Fibrinogen Level ◽  
Specific Inhibitor ◽  
Test System ◽  
Rabbit Brain ◽  
Clotting Time ◽  
Normal Plasma ◽  
Tissue Thromboplastin ◽  
Dilute Suspension

A severe hemophiliac (<1% FVIII-AHG) was transfused with FVIII concentrate and had orthopedic surgery without complication. His in vivo FVIII-AHG survival was normal (T½= 8hrs), as was his fibrinogen level, prothrombin (PT) and thrombin times. His prolonged (>100 sec.) activated partial thromboplastin time was incompletely corrected (40 sec.; normal<36 sec.) by the in vitro addition of an equal volume of pooled normal plasma, and did not become further prolonged after ½ and 2 hr. incubations at 37°C. A specific inhibitor to FVIII-AHG was not present. However, a thromboplastin inhibition test using patient plasma and progressively higher dilutions of rabbit brain thromboplastin (whole phospholipoprotein membranes) in a PT test system was positive. When either normal gel-separated platelets (GSP) or a dilute suspension of inosithin was used as a source of phospholipid, and clotting then initiated by the simultaneous addition of calcium and Russell’s viper venom (to activate FX directly), patient plasma clotting times were similar to the clotting times of normal plasma and other severe FVIII-AHG deficient plasmas. In contrast, when both normal GSP and dilutions of tissue thromboplastin reagent were added to plasma, and clotting then initiated by recalcification, patient plasma clotting time was prolonged in comparison to the clotting times of normal plasma and other FVIII-AHG deficient plasmas. This “lupus-like” anticoagulant had no effect on in vivo hemostasis, even in our patient with severe hemophilia A. The anticoagulant differs from a lupus anticoagulant recently well characterized (Thiagarajan et al., J.Clin. Invest. 66;397,1980) in that it interacts better with phospholipoprotein membranes than with free phospholipids, and the in vitro defect is not corrected by the addition of normal platelets.

scholarly journals A Comparative Study of the Effects of Heparin and a Low Molecular Weight Semi-Synthetic Heparin Analogue Upon Platelet Function

1977 ◽  
Author(s):  
R. Michalski ◽  
D. A. Lane ◽  
V. V. Kakkar
Keyword(s):  
Molecular Weight ◽  
Intravenous Injection ◽  
Platelet Function ◽  
Factor Xa ◽  
In Vitro Tests ◽  
Low Molecular Weight ◽  
Clotting Time ◽  
Induced Aggregation

We have already reported O) some in vitro and in vivo properties of a low molecular weight glycosaminoglycan polysulphate. It was found that while this semi-synthetic heparin analogue (SSHA) was virtually inactive in a number of in vitro clotting assays, following intravenous or subcutaneous injection it has a more specific anti-Xa potentiating effect than heparin. In the present communication a comparison has been made of some effects of SSHA and heparin upon platelet function. In several of the in vitro tests performed, such as their potentiating effect on ADP and adrenaline induced aggregation and their effects on the aggregation of washed platelets by Factor Xa, heparin proved to be far more potent than SSHA. It was found that after intravenous injection of both drugs, PRP samples containing comparable anti-Factor Xa activities responded differently to the addition of thrombin as SSHA barely inhibited thrombin induced aggregation. Similarly, SSHA had little effect on the dilute thrombin clotting time of plasma, following intravenous injection. Heparin and analogue were neutralised to approximately the same degree by a crude PF4 preparation, and similar transient thrombocytopenia effects were observed with both drugs.

Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

1990 ◽  
Vol 63 (02) ◽  
pp. 220-223 ◽  
Author(s):  
J Hauptmann ◽  
B Kaiser ◽  
G Nowak ◽  
J Stürzebecher ◽  
F Markwardt
Keyword(s):  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Factor Xa Inhibitors ◽  
Venous Stasis ◽  
Clotting Time ◽  
Thrombosis Model ◽  
Activity Factor ◽  
Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

Inhibition of Fibrinolysis and Fibrinogenolysis in Man: Comparison of ε-Aminocaproic Acid and Kallikrein Inhibitor

1963 ◽  
Vol 10 (01) ◽  
pp. 106-119 ◽  
Author(s):  
E Beck ◽  
R Schmutzler ◽  
F Duckert ◽  
Keyword(s):  
Aminocaproic Acid ◽  
Side Reactions ◽  
In Vitro Tests ◽  
Factor V ◽  
Clinical Use ◽  
Strong Inhibitor ◽  
Kallikrein Inhibitor ◽  
Therapeutic Possibilities

SummaryInhibitor of kallikrein and trypsin (KI) extracted from bovine parotis was compared with ε-aminocaproic acid (EACA): both substances inhibit fibrinolysis induced with streptokinase. EACA is a strong inhibitor of fibrinolysis in concentrations higher than 0, 1 mg per ml plasma. The same amount and higher concentrations are not able to inhibit completely the proteolytic-side reactions of fibrinolysis (fibrinogenolysis, diminution of factor V, rise of fibrin-polymerization-inhibitors). KI inhibits well proteolysis of plasma components in concentrations higher than 2,5 units per ml plasma. Much higher amounts of KI are needed to inhibit fibrinolysis as demonstrated by our in vivo and in vitro tests.Combination of the two substances for clinical use is suggested. Therapeutic possibilities are discussed.

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