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Sulfated beta-O4 low molecular weight lignins
Low molecular weight sulfated beta-O4 lignin (SbO4L) are potent inhibitors of coagulation with high selectivity.
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Field of the Invention
This invention is related to a class of synthetic sulfated beta-O4 low molecular weight lignins which are inexpensive to prepare and which have been found to have potent anticoagulation properties in human plasma and whole blood.
Background of the Invention
Heparin (also known as unfractionated heparin (UFH)) is extensively used in the clinic as anticoagulant.1 UFH is obtained from pig intestinal or lung mucosa and is relatively inexpensive. It is then processed to produce low molecular weight heparins (LMWHs), which are also powerful anticoagulants. Enoxaparin is one such LMWH and is obtained from UFH by chemical treatment. Other LMWHs available in the clinic are obtained from UFH either through chemical or enzymatic means.
Both UFH and LMWHs are used to treat numerous thrombotic disorders including deep-vein thrombosis (DVT), disseminated intravascular coagulations (DIC), pulmonary embolism (PE), acute myocardial infarction, unstable angina and cerebrovascular thrombosis.1-3 These are also used during surgery, organ transplantation and for extracorporeal bypass procedures. Combined the annual market of heparins is more than $8 billion within the USA alone.
Yet, both heparins have their own disadvantages. Both agents are heterogeneous mixtures of highly sulfated polysaccharide chains, which introduce a number of issues. The biggest drawback is of significant risk of internal bleeding, which might be of either minor bleed or major bleed type. UFH, and sometimes LMWH, is associated with the occurrence of thrombocytopenia in about 3% of patients.4 Osteoporosis could also arise in some patients upon prolonged heparin usage. In addition to these adverse effects, heparin usage is problematic from quality control and administration perspective. Both UFH and LMWHs possess significant structural variability, which affects their bioavailability and pharmacokinetic parameters. Thus, patient response variability is high. It is also difficult to ascertain the absence of non-heparin-like molecules in a preparation of heparin, e.g., chondroitin sulfate in heparin, because of their structural similarity and mode of preparation. Events of 2008, wherein contamination of UFH by oversulfated chondroitin sulfate led to the death of 81 people in the US, demonstrate the difficulty of quality control with these highly heterogeneous preparations.5
It would be advantageous to identify new anticoagulants which possess heparin-like activity without the heterogeneity present in UFH and LMWHs. Further, new anticoagulants, particularly which can be obtained through inexpensive route so as to compete with the cost of UFH therapy, will provide useful alternatives for a number of clinical and other applications.
Embodiments of the invention relate to sulfated beta-O4 low molecular weight lignin (SbO4L), an oligomer which may be prepared using a fully chemical system and possessing highly specific and potent thrombin inhibitory activity. SbO4L has excellent human plasma and blood anticoagulant potential. SbO4L is an oligomeric lignin molecule that contains only one type of inter-monomeric residue linkage i.e. the β-O-4 linkage. This reduces heterogeneity dramatically so much so that the agent can be assessed for purity using, for example, standard chromatographic tools. The anticoagulant is easily prepared in few simple steps, and thus the new anticoagulant may be inexpensive. Further, industrial scale preparation of SbO4Ls should be possible, thereby increasing its utility. The new anticoagulants described herein should have significant clinical applications.
According to embodiments of the invention, there is described a generic sulfated beta-O4 lignin (SbO4L) scaffold for a family of lignin compounds. These lignin compounds are of low molecular weight (e.g., Molecular Weight ranging from 5000 to 15000 (or 8000 to 12000)). SbO4L has the general chemical structure:
where Y=SO3M (M=Na+, Ca2+, NH4+ and other equivalent positively charged ions) or H;
n can be 0-50 (e.g., 4-6, 5-10, 5-25, 10-25, 15-25, 10-20, 15-50 as well as other ranges therebetween);
R2, R3, R5, and R6 can be hydrogen (—H), C1-10 linear alkyl (e.g., —CH3, —CH2CH3, etc.), C1-10 branched alkyl (e.g., —CH(CH3)2, —CH2CH(CH3)2, etc.), C1-10 and O1-10 oxyalkyl (e.g., —OCH3, —OCH2OCH3, etc.), electron-donating (e.g., —OH, —OR (R═C1-10 alkyl, aryl, allyl, and benzyl), —NH2, —NHR (R═C1-10 alkyl, aryl, allyl, and benzyl), electron-withdrawing (e.g., —NO2, —SO3H, —SO3M (M=Na+, Ca2+, NH4+ and other equivalent positively charged ions) or halogens (—F, —Br, —Cl and —I), and can be the same or different; and
chiral centers denoted by (*) are either R or S, and can be the same or different.
Examples of SbO4L ligands include tetrameric-hexameric molecules and decameric-pentadecameric molecules represented by the following generalized structures:
In each of the two exemplary SbO4L families of compounds above, Y is preferably H or SO3Na.
In particular embodiments, mixtures are provided wherein the mixtures contain two or more different sulfated lignin scaffolds, each of which has the generic SbO4L scaffold. For example, the two or more different sulfated lignin scaffolds could have different sizes or have different constituents (i.e., R2, R3, R5 or R6 differ between successive aromatic units), etc. (e.g., different lignins in a mixture could have varying numbers of repeats (n) or one or more different substitutions (e.g., different charged ions, different moieties, such as aryl, alkyl, allyl, oxyalkyl, etc.).
In other embodiments, compositions are provided which including one or more carriers or matrices mixed with one or more sulfated lignins, wherein at least one of the sulfated lignins has the generic SbO4L scaffold. In some compositions, mixtures of two or more different sulfated lignins, where each sulfated lignin has the generic SbO4L scaffold. In some compositions, at least one carrier which is a pharmaceutically acceptable liquid (e.g., saline, oil, water (deionized, distilled, etc.), etc.) is included in the composition. In some compositions, at least one matrix which is a pharmaceutically acceptable sold (e.g. cornstarch, etc.) is included in the composition.
In still other embodiments, methods of producing sulfated lignins with the generic SbO4L scaffold are disclosed. In particular embodiments, a simple three step process of polymerization, reduction, and sulfation is used.
In further embodiments, methods of using sulfated lignins with the generic SbO4L scaffold are disclosed. Exemplary embodiments include using the sulfated lignins, or mixtures, or compositions thereof as an anticoagulant (e.g., through direct, allosteric inhibition of clotting enzymes) and in any application where UFH and LMW may be employed.
The invention is related to a family of low molecular weight lignin compounds (e.g., MW ranging from 5000 to 15000) defined by a generic sulfated beta-O4 lignin (SbO4L) scaffold having the general chemical structure:
where Y=SO3M (M=Na+, Ca2+, NH4+ and other equivalent positively charged ions) or H;
n can be 0-50 (e.g., 4-6, 5-10, 5-25, 10-25, 15-25, 10-20, 15-50 as well as other ranges therebetween);
R2, R3, R5, and R6 can be hydrogen (—H), C1-10 linear alkyl (e.g., —CH3, —CH2CH3, etc.), C1-10 branched alkyl (e.g., —CH(CH3)2, —CH2CH(CH3)2, etc.), C1-10 and O1-10 oxyalkyl (e.g., —OCH3, —OCH2OCH3, etc.), electron-donating (e.g., —OH, —OR (R═C1-10 alkyl, aryl, allyl, and benzyl), —NH2, —NHR (R═C1-10 alkyl, aryl, allyl, and benzyl), electron-withdrawing (e.g., —NO2, —SO3H, —SO3M (M=Na+, Ca2+, NH4+ and other equivalent positively charged ions) or halogens (—F, —Br, —Cl and —I), and can be the same or different; and
chiral centers denoted by (*) are either R or S, and can be the same or different.
The low molecular weight sulfated beta-O4 lignin(s) (SbO4L) are oligomer(s) which can be prepared using a fully chemical system and they possess highly specific and potent thrombin inhibitory activity. Hence, SbO4L lignins have excellent human plasma and blood anticoagulant potential. A defining feature is that SbO4L is an oligomeric lignin molecule that contains only one type of inter-monomeric residue linkage i.e. the β-O-4 linkage. This reduces heterogeneity dramatically, so much so that the agent can be assessed for purity using standard chromatographic tools. The anticoagulant is easily prepared in few simple steps, suggesting that the new anticoagulant is likely to be inexpensive. Further, industrial scale preparation of SbO4Ls is possible, thereby increasing its utility.
The synthesis of the SbO4Ls can be achieved in three easy steps using, for example, alpha-bromo-1,3-ketoester and aryl phenol. The first step involves the polymerization step utilizing a base such as potassium carbonate.
Example of Polymerization: In a flask containing the monomer ethyl 2-bromo-3-(4-hydroxy-3-methoxyphenyl)-3-oxo-propanoate (0.97 g) (1) (or other appropriate monomer) in anhydrous DMF (5 mL), anhydrous K2CO3 (0.58 g) was added and stirred under nitrogen. After 24 h the reaction mixture was poured onto ice-water mixture (120 ml) and the pH adjusted to ˜2.5 with 2M HCl. The precipitated polymer (2) was filtered, washed with water and lyophilized to remove moisture. In the Example of Polymerization R1=—COOEt.
NMR characterization showed the following corresponding broad peaks for 1H-NMR (DMSO-d6): 7.46-7.78 (C2-H and C5-H), 6.91-7.14 (Cβ-H), 6.52-6.66 (C6-H), 4.19-4.20 (—OCH2CH3), 3.79-3.81 (—OCH3), 1.12-1.22 (—OCH2CH3).
13C-NMR (DMSO-d6) 188.53, 189.08 (Cα), 165.81, 166.02 (Cγ), 153.16, 150.93, 149.48, 149.01, 147.64, 128.33, 125.51, 124.75, 123.73, 115.17, 114.08, 113.91, 113.09, 112.51, 112.30 (aromatic carbons), 78.54, 78.37 (Cβ), 61.80, 61.67 (—OCH2CH3), 55.78, 55.58 (—OCH3), 13.77 (—OCH2CH3).
After polymerization, the second step is a reduction of the polymer which can be achieved with methanol.
Example of Reduction: The solid (600 mg) (2) was suspended in methanol (10 ml) at 50° C. followed by careful addition of NaBH4 (683 mg) while maintaining 50° C. The mixture gradually turned into a clear solution, which was stirred for 24 h. The solution was then neutralized with acetic acid and poured into 0.5M HCl (200 mL) to precipitate the reduced polymer, which was isolated by centrifugation, washed with water and lyophilized. Dissolution and preferential precipitation using 1,4-dioxane and diethyl ether helped remove low molecular weight chains of the polymer and precipitate the high molecular weight chains. The precipitate (3) was filtered and dried using vacuum.
NMR characterization showed the following corresponding broad peaks for 1H-NMR (DMSO-d6): 4.75 (Cα-H), 4.28 (Cβ-H), 3.20-3.71 (Cγ-H and —OCH3), 7.02 (C2-H), 6.94 (C5-H), 6.85 (C6-H).
13C-NMR (DMSO-d6): 70.8, 71.4 (Cα), 83.6, 84.4 (Cβ), 59.8, 59.9 (Cγ), 55.4 (—OCH3), 134.7-135.0 (C1), 111.3-111.7 (C2), 148.9 (C3), 146.7-147.0 (C4), 114.5-115.0 (C5), 119.4-119.7 (C6).
The final step is sulfation of the free hydroxy groups.
Example of Sulfation: The reduced polymer (50 mg) (3) so obtained was made to react with triethylamine-sulfur trioxide complex (200 mg) in anhydrous DMF (5 ml) at 65° C. for 24 h under reflux. Following the reaction, 30% (w/v) aqueous sodium acetate (35 ml) was added and the mixture stirred overnight. The solution was then poured into ice-cold ethanol (100 ml) to precipitate crude SbO4L LMWL, which was filtered and washed twice with ice-cold ethanol (10 ml each). The recovered solid was desalted using FloatALyzer G2 (Spectrum Labs) dialysis tubes (MWCO 0.1-0.5 KDa). Lyophilization of the dialyzed solution gave SbO4L (4). NMR characterization showed the following corresponding broad peaks for 1H-NMR (DMSO-d6): 6.77 (aromatic protons), 3.47-4.27 (Cα-H, Cβ-H, Cγ-H, —OCH3).
13C-NMR could not give sufficient signal even at high concentrations of the sample, possibly due to the presence of a wide variety of sulfation patterns and molecules of different chain lengths.
The final step in the exemplary synthesis procedure is sulfation of the free hydroxy groups. Purification of the final product can be performed by either aqueous dialysis or by ultrafiltration. The SbO4L so synthesized was studied for thrombin, factor Xa, factor IXa, factor XIa, factor VIIa and factor XIIa inhibition and found to exhibit potent inhibition of thrombin alone. It prevented human plasma and whole blood clotting at pharmaceutically relevant concentrations and was active in in vivo models.
The molecular weight of the SbO4Ls is low and can range from 5000-15000 Dalton and may be more precisely controlled to e.g., 8000 to 12,000 Da, or 9000±500 Da. For exemplary purposes with regarding molecular weight measurements, gel permeation chromatography (GPC) of SbO4L using Phenogel 5 micron (Phenomenex, Torrance, Calif., 7.6 mm i.d.×300 mm) column and 0.1 N NaOH mobile phase was performed at a constant flow rate of 0.7 mL/min and gave a characteristic broad elution profile by monitoring absorbance at 254 nm. Polystyrene standards of different molecular weights and ferulic acid were used for calibration purposes. The relationship between logarithm of the molecular weight and the elution volume (V) of the standards was found to be linear with a correlation co-efficient of 0.99. The SbO4L chromatogram was sliced into 1000 time periods providing 1000 average molecular weights with their corresponding absorbances. These values were used to calculate MW, MN, and MP values. The molecular weight of the final sulfated lignins were measured using aqueous phase GPC-HPLC and found to have a number average molecular weight MN of 9,200 Da and weight average molecular weight MW of 12,300 Da. This procedure is similar to that used earlier for sulfated low molecular weight lignins.
As demonstrated below, the SbO4L compounds, or mixtures thereof, may be useful as an anticoagulant either in vitro, ex vivo or in vivo. They appear to act direct, allosteric inhibition of clotting enzymes. These SbO4L lignin compounds may be combined with a pharmaceutical carrier. In some formulations, the SbO4L lignins can be the same or different. In some formulations, the SbO4L lignins can be combined with other lignins or other selected materials. Suitable pharmaceutically acceptable carriers are known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration for in vivo applications, or for addition to blood or plasma in in vivo, in vitro, and ex vivo applications may also be prepared. The preparation (e.g., a composition containing one or more SbO4L compounds) may also be emulsified. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of SbO4L in the formulations may vary. However, in general, the amount in the formulations will be from about 1 to about 99% by weight, (e.g., 5% or 10% to 20%, 30%, 40%, 50%, or 60% of one or more SbO4L compounds with the remainder being pharmaceutical carriers, excipients and/or other ingredients).
The SbO4L compositions can be added directly to blood or plasma in an in vitro or ex vivo application.
For in vivo applications, the SbO4L compositions (preparations) may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, inhalation, orally, intranasally, by ingestion of a food product containing the antagonist, topically, as eye drops, via sprays, etc. In preferred anticoagulant embodiments, the mode of administration may be intravenous provisioning or topical application. In addition, the compositions may be administered in conjunction with other treatment modalities such as other medicaments, other types of therapy, and the like.
The amount of SbO4L that is administered to an individual in an in vivo application (who is usually a mammal, typically a human) will vary based on several factors, as will be understood by those of skill in the art. For example, the dose and frequency of administration may vary according to the gender, age, weight, general physical condition, ethnic background, etc. of the individual, as well as whether or not the individual has other diseases or conditions that might impinge on the treatment. Generally, the dose will be in the range of from about 0.01 to about 10000 mg/kg of body weight (e.g., 1 to 50, 100, 250 or 500 mg/kg, etc.).
Exemplary applications of the SbO4L lignins include, but are not limited to 1) as anticoagulant and/or antithrombotic agents in cardiovascular disease therapy; 2) as anti-HSV-1, anti-HSV-2, and anti-HIV-1 agents; 3) as agents against other viruses include dengue, varicella, etc.; 4) as regulators of angiogenesis with implications in cancer; 5) regulator of stem cell growth (either inhibition or proliferation). Their anticoagulant activity can be mediated by already available antidote protamine which is FDA approved for heparin toxicity. SbO4L compounds are less heterogeneous than heparin and low molecular weight heparins. SbO4L compounds are potent inhibitors of coagulation with high selectivity. Mechanistically, they possess novel mode of action and thus are radically different from all known anticoagulants. The SbO4L are readily synthesized, and are not isolated from animal sources and hence less prone to prion borne diseases.
A Non-Saccharide, Synthetic Heparin Mimetic that Exhibits Potent Plasma and Blood Anticoagulation Through Selective, Direct and Allosteric Thrombin Inhibition
Sulfated low molecular weight (LMW) lignins which are non-saccharide mimetics of LMW heparins exhibit potent plasma and blood anticoagulation through direct, allosteric inhibition of clotting enzymes. Sulfated β-O4 lignin (SbO4L) is an advanced molecule that is easily synthesized in only three steps and is homogeneous in terms of inter-monomer linkages. SbO4L was found to potently inhibit human thrombin and plasmin (IC50=15-34 nM), while related coagulation factors or heparin-binding serine proteases were inhibited with orders of magnitude weaker potency. Plasma antithrombin did not affect SbO4L inhibition of thrombin, while activation of protein C by thrombin-thrombomodulin complex was inhibited with 20-times poorer potency by SbO4L. Kinetic studies showed reduction in maximal rate of substrate hydrolysis, but not in Michaelis constant, indicating a direct allosteric mechanism of action. Competitive inhibition studies suggested that SbO4L interacted with heparin-binding exosite 2 of thrombin. Human plasma and whole blood coagulation assays showed that SbO4L's anticoagulation effect was comparable to that observed with a clinically used LMW heparin. Protamine rapidly and quantitatively reversed SbO4L inhibition of thrombin. Overall, SbO4L is a non-polysaccharide, non-animal derived, very readily synthesizable, thrombin-selective, direct allosteric anticoagulant that can truly challenge the wide-spread clinical use of heparins.
Thrombotic disorders such as pulmonary embolism, deep vein thrombosis, and disseminated intravascular coagulation are a major cause of mortality in humans. Heparin and warfarin, two key anticoagulants that treat pro-thrombotic conditions, were discovered in the first half of the 20th century and are still used in essentially the same form.1 Yet, both suffer from serious bleeding risks and adverse reactions.1-5 Low molecular weight (LMW) heparins have a better safety profile, but are not devoid of bleeding events.4,6 Clinical data collected over the past decade shows that newer agents introduced in the clinic including bivalirudin, argatroban, and fondaparinux do not significantly improve anticoagulation therapy over heparins,7-9 while initial indications with the recently introduced drug, dabigatran, are that it too suffers from bleeding issues.10,11 Ximelagatran, introduced in 2003, had to be quickly withdrawn because of significant hepatotoxicity. Initial reports with rivaroxaban, introduced in Europe in 2008 and in the US in July 2011, indicate significant benefit with some bleeding risks, but a conclusive statement on its safety record will take time.12,13
Mechanistically, anticoagulants can be either active site- or allosteric site-targeting agents. Examples of the enzyme active site inhibitor class include argatroban, melagatran, rivaroxaban, DX9065a, apixaban, and numerous others in various stages of clinical trials. In contrast, no molecule has been introduced in the clinic that inhibits a coagulation enzyme on an exclusively allosteric basis.
Allosteric regulation of coagulation enzyme's function offers a couple of major advantages. Allosterism offers the possibility of fine control over an enzyme's activity. Whereas the efficacy of competitive inhibitors is typically 100%, that for allosteric inhibitors may be tunable so as to achieve 'regulation', i.e., less than quantitative inhibition.14,15 In principle, allosteric thrombin regulation can strike a delicate balance between pro- and anti-coagulant activities. Allosteric regulation can also better realize specificity of function. Coagulation enzymes are all trypsin-like, homologous enzymes with considerable similarity in their active site geometry.16 In contrast, significantly greater differences are found in their exosite geometries. For example, exosite 2 of thrombin, which binds heparin, bears some homology to a corresponding site in factor Xa, but display little correspondence to similar sites in factors IXa and XIa.17 In effect, allosteric regulation promises exquisite control over both specificity of recognition and efficacy of inhibition.
Recently, our laboratory designed the first few examples of exclusively allosteric coagulation enzyme inhibitors. These include the sulfated LMW lignins and sulfated benzofurans.17-22 Sulfated LMW lignins were originally designed to mimic the allosteric interaction of heparin with antithrombin, a key regulator of coagulation.23,24 Yet, detailed studies pointed to direct inhibition of thrombin, factor Xa and factor XIa as the major mechanism of action.17,22,25 Interestingly, these molecules were found to bind in the heparin-binding site of these enzymes and induce allosteric inhibition.22,25
Sulfated LMW lignins are similar to unfractionated heparin (UFH) or LMW heparins in terms of structural polydispersity and heterogeneity (
In this Example, we report on the development of a sulfated β-O4 lignin (SbO4L), a chemically synthesized lignin, which exhibits highly selective and potent inhibition of human thrombin (and plasmin). SbO4L is homogeneous with regard to its inter-monomer linkages. It inhibits thrombin through an allosteric process by binding in exosite 2. Human whole blood studies demonstrate that SbO4L-based anticoagulation that is comparable to that induced by enoxaparin. Most importantly, SbO4L is synthesized in a few simple steps from readily available small molecules, which bodes well for a relatively inexpensive alternative to UFH and LMW heparin. Additionally, SbO4L inhibition of thrombin is quantitatively and rapidly reversed by protamine, which should greatly increases confidence in its usage. Thus, this Example describes a family of completely synthetic, non-polysaccharide, non-animal derived, thrombin-selective, allosteric anticoagulants which may be used in treating human and animal subjects.
Experimental Procedures
Proteins and Chemicals.
Human proteases were from either Haematologic Technologies (Essex Junction, Vt.), Sigma-Aldrich (St. Louis, Mo.), or Elastin Products Company (Owensville, Mo.). Chromogenic substrates were obtained either from Sekisui Diagnostics (Stamford, Conn.), Sigma-Aldrich or Diapharma (West Chester, Ohio). HirP, a Tyr63-sulfated hirudin-(54-65) peptide labeled with 5-(carboxy)fluorescein, i.e., [5F]-Hir[54-65](SO3−), was from the Bock laboratory.26 Monomer used in SbO4L preparation was synthesized using literature protocol.27 Plasma and whole blood clotting reagents were from Fisher Diagnostics (Middletown, Va.) or Haemoscope Corporation (Niles, Ill.).
Chemical Synthesis of Sulfated β-O4 Lignin (SbO4L).
The synthesis of SbO4L was developed from reported polymerization and sulfation strategies. Briefly, K2CO3 and ethyl 2-bromo-3-(4-hydroxy-3-methoxyphenyl)-3-oxo-propanoate were stirred in anhydrous DMF under nitrogen for 24 h, poured into ice-water mixture and the pH adjusted to ˜2.5 with 2M HCl. The precipitated polymer was filtered and lyophilized. The solid was suspended in methanol at 50° C. and NaBH4 carefully added. After 24 h, the solution was neutralized with acetic acid, poured into 0.5M HCl and precipitated polymer was isolated by centrifugation. The solid was then dissolved in 1,4-dioxane and high molecular weight chains precipitated in diethyl ether. The reduced polymer was sulfated with triethylamine-sulfur trioxide complex in anhydrous DMF at 65° C. for 24 h,30,31 dissolved in 30% (w/v) aqueous sodium acetate and poured into ice-cold ethanol to precipitate crude SbO4L. The recovered solid was desalted using FloatALyzer G2 (Spectrum Labs) dialysis tubes (MWCO 0.1-0.5 kDa) and lyophilized.
Molecular Weights of SbO4L.
The number—(MN), weight—(MW), and peak—(MP) average molecular weights of SbO4L were measured as described earlier.23 Briefly, gel permeation chromatography (GPC) of SbO4L was performed on a Phenogel 5μ column (Phenomenex, Torrance, Calif.) using 0.1 N NaOH mobile phase flowing at 0.7 ml/min with detection at 254 nm. The SbO4L chromatogram was sliced into 1000 time periods providing 1000 average molecular weights with their corresponding absorbances, which were used to calculate MW, MN, and MP values.23
Direct Inhibition of Proteases.
Inhibition of coagulation enzymes (thrombin, factors Xa, IXa, XIa and VIIa) by SbO4L was measured using chromogenic substrate hydrolysis assays in standard manual 1 ml cuvettes, as described previously,17,21,22,25 while inhibition of other proteases was measured using a 96-well microplate format. This protocol was also adopted for studying thrombin inhibition in the presence of antithrombin or in the presence of serum albumin. The buffers, conditions and chromogenic substrates used in these experiments were derived from the literature. Briefly, a SbO4L in an appropriate buffer was incubated with a target protease for 10 min, followed by addition of chromogenic substrate and rapid measurement of the initial rate of substrate hydrolysis (A405). The ratio of the initial rate in the presence and absence of SbO4L gave the residual protease activity at each inhibitor concentration from which the IC50 was calculated using logistic equation 1.
In this equation, Y is the residual protease activity; YM and Y0 are the maximum and minimum residual activities, respectively; IC50 is the concentration of the inhibitor that results in 50% inhibition of enzyme activity; and HS is the Hill slope.
Inhibition of Thrombin-Thrombomodulin-Mediated Activation of Protein C by SbO4L.
The protocol for measuring the activation of protein C by thrombin-rTM complex essentially followed the literature.32 Briefly, 5 μl of SbO4L (2.3 ng/ml to 2.3 mg/ml) was added to 70 μl of 50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, and 1 mM CaCl2 at 37° C., followed by addition of 5 μl of thrombin (240 nM) and 5 μl of rTM (400 nM). To this mixture, 5 μl of protein C (10 μM) was added followed by incubation for another 10 min. At the end of this period, APC generation was quenched with 5 μl of 10 mM EDTA and 16 μM argatroban, and quantified from the initial rate of hydrolysis of 400 μM S-2366. The apparent IC50 of SbO4L inhibition of activation of protein C was calculated using equation 1.
Michaelis-Menten Kinetics and Competitive Binding Studies.
The kinetics of substrate hydrolysis by thrombin in the presence of SbO4L and competitive studies with exosite 1 and exosite 2 ligands followed our protocols for sulfated LMW lignins,17-19,22
Plasma and Whole Blood Clotting Assays.
The protocol for human plasma activated partial thromboplastin (APTT) and prothrombin time (PT), and human whole blood thromboelastography (TEG) and hemostasis analysis (HAS) in the presence and absence of SbO4L followed our earlier studies with sulfated LMW lignins.18,19,21,23
Reversal of SbO4L Activity by Protamine.
Protamine (4.9 μg/l to 14.8 g/l was added to a solution of SbO4L-thrombin complex in a 20 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, 2.5 mM CaCl2 and 0.1% PEG8000 at 37° C. followed by measuring the residual thrombin activity from substrate hydrolysis, as described above. In the absence of protamine, SbO4L gave ˜50% inhibition of thrombin at 37° C. Protamine alone did not affect the activity of thrombin as assessed by appropriate controls.
Synthesis and Characterization of SbO4L.
Of the several inter-residue linkages present in lignins, the β-5 and β-O4 linkages are most abundant.33 Earlier work had shown that sulfated LMW lignins devoid of the β-5 linkage, and hence enriched in β-O4 linkage, generated significant anticoagulant activity.22,23 Based on this, we reasoned that a lignin containing only β-O4 linkage may exhibit much higher anticoagulation potential. Hence, we resorted to a wholly chemical process of alkaline polymerization of ethyl 2-bromo-3-(4-hydroxy-3-methoxyphenyl)-3-oxo-propanoate followed by sodium borohydride reduction and sulfation using triethylamine-sulfur trioxide complex (
The MN, MW and MP of SbO4L were measured using aqueous GPC-HPLC, as described earlier,23 and found to be 9200, 12300, and 9100, respectively. This implies that on an average SbO4L oligomer is nearly 23 residues long. In comparison, an average full-length heparin chain is ˜50 saccharides in length, whereas an average LMW heparin chain has 16 residues. The GPC-HPLC chromatograms also indicated absence of species with molecular weight less than 1000 suggesting an enriched preparation of longer oligomers that constituted composition greater than 95%. The polydispersity, i.e., the ratio of MW to MN, of SbO4L was found to be 1.336, which is similar to that of UFH used in the clinic. The sulfation density calculated on the basis of the difference in MW between SbO4L and its unsulfated precursor was ˜2 sulfate groups per monomer. Elemental analysis measurements indicated a composition of C, 35.39; H, 4.15; and S, 11.54; which match a hypothetical SbO4L chain of 23 residues containing 27 sulfate groups and calculated composition of C, 35.20; H, 4.02; S, 11.08. This indicates an average of 1.2 sulfate groups per monomer. These measurements compare favorably with that of UFH and LMW heparins, which show an average of ˜1.6 anionic groups per monomer. Additional purity and consistency of SbO4L preparation studies were performed using reversed-phase ion pairing UPLC-MS, and in vitro thrombin inhibition tests on three independent batches of synthetic SbO4L. The three batches demonstrated essentially identical UPLC-MS fingerprints and indistinguishable IC50 for thrombin inhibition. Thus overall, the elemental analysis measurements in combination with UPLC-MS fingerprints indicate the presence of uniform β-O4 linkage in SbO4L preparation in high purity (>95%) in comparison to sulfated LMW lignins studied earlier.
SbO4L is a Selective, Direct Inhibitor of Human Thrombin.
The inhibition of coagulation factors IIa, Xa, IXa, XIa and VIIa was studied using spectrophotometric measurement of the residual protease activity in the presence of varying levels of SbO4L.
SbO4L inhibits thrombin with an IC50 of 0.17 □g/ml, which corresponds to ˜14 nM (Table 1). Intrinsic pathway proteases factors Xa and XIa were also inhibited by SbO4L, however the IC50 values were in the range of 90-500 μg/ml corresponding to 10-55 μM. With regard to factor IXa, essentially no inhibition was noticeable at concentrations as high as 28.7 μg/ml (
With regard to the extrinsic pathway, SbO4L was found to inhibit factor VIIa with an IC50 of 0.54 μg/ml (
To assess the selectivity features of SbO4L in more detail, we studied a small group of heparin-binding serine proteases including cathepsin G, HLE, kallikrein, PPE, APC, plasmin, chymotrypsin, and trypsin. The inhibition of these proteases by sulfated LMW lignins, parent molecules from which SbO4L was designed, was studied earlier.25 The activity of each protease was measured in a spectrophotometric assay in the presence of SbO4L at 0.37 (
SbO4L Inhibition of Thrombin is Unaffected by the Presence of Antithrombin.
Polymeric heparin is a powerful antagonist of thrombin because of its ability to bind plasma antithrombin and bridge with thrombin.1,34 In contrast, sulfated LMW lignins were found to bind the serpin with reasonable affinity, yet loose direct inhibition potential.24 To assess the influence of antithrombin on SbO4L activity, direct inhibition of thrombin was studied in the presence of 200 nM serpin under otherwise identical conditions. A decrease in thrombin activity with increasing SbO4L levels was observed, which was identical to that measured in the absence of the serpin, except for the differences in maximal (YM) and minimal (YO) residual thrombin activities. The YM and YO values decreased by 35% and 23%, respectively, due to the reaction of thrombin with antithrombin. However these changes did not affect SbO4L inhibition of thrombin (IC50=0.2 and 0.17 μg/ml with and without antithrombin, respectively (Table 1)). From this result it can be inferred that antithrombin does not retard or stimulate SbO4L's inhibitory activity. This is a significant point of variance from the parent sulfated LMW lignins, for which the serpin-mediated pathway was found to be a competing side reaction.24 The results highlight an interesting structural aspect that a specific scaffold, i.e., the β-O4-linked scaffold, of the many possible in sulfated LMW lignins does not appear to bind the serpin with high affinity, which improves mechanistic selectivity.
SbO4L Inhibits Activation of Protein C by Thrombin-rTM Complex.
Thrombin's procoagulant activity is drastically modulated by cell surface TM, which transforms it into an anticoagulant protease with specificity for protein C.35,36 To assess whether TM binding to thrombin alters the activity of SbO4L, we studied the efficacy of protein C activation. The level of proteolytically active protein C formed by thrombin-rTM complex can be measured spectrometrically through hydrolysis of S-236632 following suppression of thrombin's native proteolytic activity using argatroban (˜100×K1)37 as its specific inhibitor. Measurement of APC levels in the presence of varying SbO4L levels led to a sigmoidal profile on a semi-log plot (
SbO4L is an Allosteric Inhibitor of Thrombin.
To understand the basis of thrombin inhibition, we measured the kinetics of Spectrozyme TH hydrolysis at pH 7.4 in the presence for SbO4L. Plots of the initial rates versus substrate concentration were hyperbolic, as expected (
Allostery Arises from Binding in Exosite 2 of Thrombin and not in Exosite 1.
To decipher the origin of allostery, we measured the effect of HirP, a hirudin-based dodecapeptide, on the SbO4L inhibition of thrombin. Earlier work has shown that HirP binds in exosite 1 with an affinity of 28 nM and increases the catalytic efficiency of thrombin.26 Thus, thrombin inhibition by SbO4L was studied in the presence of the exosite 1 competitor (
These results imply that the interaction of HirP with thrombin does not affect SbO4L inhibition to a significant extent suggesting that the allosteric inhibitor does not engage exosite 1.
To assess whether SbO4L binds in exosite 2 of thrombin, we studied competition with UFH.
The efficacy of competition can be gauged by using the Dixon-Webb relationship (Eq. 2), which estimates the ideality of competition between two ligands. In this equation, KUFH is the dissociation constant of thrombin-UFH complex, which was measured to be 15.6±3.1 μM under otherwise identical conditions. Analysis using equation 2 showed that the observed IC50 was equivalent to that predicted on the basis of ideal competition. For example at 100 μM UFH, the predicted IC50 is 1.1 μg/ml, while the observed IC50 was 1.25 μg/ml. Thus, SbO4L binds in or near anion-binding exosite 2.
SbO4L is a Potent Anticoagulant in Human Plasma.
Prothrombin (PT) and activated partial thromboplastin times (APTT) are traditional measures of the anticoagulation state of human plasma.
Despite this high potency (comparable to enoxaparin), SbO4L displays a loss of ˜100-340-fold in potency in human plasma from that in in vitro enzyme systems. To assess the basis for this difference, we studied the effect of serum albumin on the effectiveness of SbO4L. FIG. 6B shows the change in relative thrombin activity in the presence of fixed concentration of SbO4L and varying concentrations of bovine serum albumin (BSA), a surrogate for its human counterpart. In the absence of BSA, thrombin's hydrolytic activity was 22% ([SbO4L]=0.42 μg/ml), which was found to increase to 56, 59 and 72% in the presence of 1, 2.5 and 10 mg/dl BSA, respectively. This suggests that the presence of BSA results in a significant drop in inhibitor potency probably arising from non-specific sequestering of SbO4L. Interestingly, a maximal thrombin activity of ˜75% is reached suggesting that some SbO4L remains free, and therefore inhibitory, at high enough plasma albumin concentration.
SbO4L is a Potent Anticoagulant of Human Whole Blood as Measured by Thromboelastography (TEG®).
To evaluate SbO4L as an anticoagulant in whole blood, we employed TEG®, which is quite often used to monitor anticoagulation therapy with LMW heparins.21 TEG® assesses the nature of physical forces within a clot, which are dramatically affected by the presence of an anticoagulant in blood. In a nutshell, the clot formation in TEG® is recorded as a force transduced on a pin at the center of a blood-containing cup. Several parameters are evaluated from this force measurement including maximum amplitude (MA), the shear elastic modulus (G), the reaction time (R) and the angle α. MA and G are measures of clot stiffness, while R and α are measures of the rate of clotting.
Whole Blood Anticoagulation Potency of SbO4L by Hemostasis Analysis (HAS™).
To further assess the whole blood anticoagulant potential of SbO4L, we utilized HAS™, which evaluates platelet contribution to clot formation (
SbO4L affects PCF and CEM in a dose-dependent manner (
SbO4L Anticoagulation can be Reversed by Protamine.
A major advantage of heparin therapy is its amenability to protamine-based reversal. It is also one of the reasons why fondaparinux continues to suffer because its iatrogenic bleeding is difficult to reverse rapidly. To test whether SbO4L inhibition of thrombin can be reversed, we studied the recovery of thrombin activity following successive introduction of protamine.
In Vivo Assays.
Two mouse models were used to analyze the anticoagulant potential of SbO4L.
FeCl3 Carotid Artery Thrombosis Model.
The ferric chloride induced injury model is commonly used to analyze the anticoagulant potential of several compounds in mice. Wild type C57B1/6 mice were anesthetized with 50 mg/kg intraperitoneal (IP) pentobarbital. SbO4L (0, 100, 300, 500 or 1000 μg in 100μ phosphate buffered saline (PBS)) was infused into the right internal jugular vein. Five minutes after infusion the right common carotid artery was exposed and fitted with a Doppler flow probe. Thrombus formation was induced by applying two 1×1.5 mm filter papers saturated with FeCl3 (3.5% solution) to opposite sides of the artery for three minutes. After washing the site of injury with PBS, flow was monitored for thirty minutes. Mice were sacrificed by pentobarbital overdose after conclusion of the experiment while under anesthesia.
Rose Bengal-Laser Injury Carotid Artery Thrombosis Model.
In vivo testing using Rose Bengal thrombosis model is another recognized mouse model used to perform anticoagulant assays. Mice were anesthetized as discussed above and 500 μg of SBO4L in 100 μl PBS was infused into the right internal jugular vein. Five minutes after infusion, Rose Bengal (75 mg/kg) was infused through the internal jugular vein, and the carotid artery was illuminated with a 1.5 mW 540 nm laser (Melles Griot, Carlsbad, Calif.) positioned 6 cm from the artery. Flow was monitored for 1.20 minutes. Mice were sacrificed by pentobarbital overdosed after conclusion of the experiment while under anesthesia. The laser light converts the dye to free radicals which result in vessel occlusion with a platelet rich thrombus in 44.7±6.5 minutes in vehicle controls. In mice treated with SbO4L, occlusion occurred in 76±21.3 minutes, demonstrating significant anticoagulant activity.
Despite a massive effort of the past 30 years it has been difficult to find a truly viable alternative to UFH and LMW heparins. The sulfated polysaccharides are very good at resolving thrombotic disorders, while being fairly inexpensive. Poly- or oligo-saccharide variants, e.g., idraparinux and its biotinylated forms,39,40 fondaparinux conjoined to a direct thrombin inhibitor,41 and heparin octasaccharides,42 continue to be developed because of the high anticoagulant efficiency achievable with the saccharide scaffold. Yet, each variant carries a major synthetic burden that cannot be expected to match the much lower cost effectiveness of the heparins. A fundamental challenge, therefore, is to design an anticoagulant that effectively challenges heparins by affording high potency and high selectivity, promising reduced adverse effects, while being very easy to produce.
SbO4L was designed so as to achieve this objective. SbO4L can be synthesized in high yields in only three steps from an appropriately functionalized monomer. This protocol is significantly more efficient than the dozens of steps used in the synthesis of fondaparinux-related anticoagulants39-42 as well as in the synthesis of active site directed anticoagulants, e.g., dabigatran, apixaban or rivaroxaban. In fact, the three-step protocol can be completed in a week using a simple synthetic set up, which is expected to put forth a highly cost effective solution to heparins. Process scale up should be possible. The chemistry used in the synthesis of SbO4L ensures that the oligomer is homogeneous with regard to inter-monomeric linkages. This greatly reduces the structural complexity in SbO4L in comparison to its precursor, sulfated LMW lignin,17,19,21-25 as well as oligomeric heparins.1,34
SbO4L is a highly potent anticoagulant in vitro and ex vivo. SbO4L inhibits thrombin (and plasmin) with high selectivity, while inhibiting factors IXa, Xa and XIa with a potency that is orders of magnitude lower. Factor VIIa-TF complex, the physiologic extrinsic pathway initiator, is inhibited with ˜10-fold lower potency, while plasma antithrombin does not affect direct action of SbO4L. Competing macromolecule TM, which alters the substrate specificity of thrombin, is likely to be functional only at ˜20-times higher concentrations of SbO4L (Table 1).
The in vitro potency against thrombin (0.17 μg/ml) changes to ˜20 μg/ml for doubling APTT and ˜80 μg/ml for effective anticoagulation of human whole blood. The primary reason for this significant drop in anticoagulant effect in plasma and blood is likely to be non-specific binding to serum albumin. This is not unusual as nearly all drugs bind to serum proteins, especially albumin. In fact, most agents exhibit significant protein binding resulting in considerable loss in effective potency. Rather binding to serum albumin may actually be an advantage because of the possibility of a slow release mechanism. On weight basis, ˜3 μg/ml enoxaparin induces a blood anticoagulation equivalent to ˜80 μg/ml SbO4L suggesting that the non-saccharide anticoagulant is likely to be nearly 20-30-fold weaker in clinical efficiency. This is not necessarily a disadvantage because the more important parameter is the ratio of potency to adverse effects (bleeding, thrombocytopenia, hepatotoxicity, etc.) in vivo.
SbO4L inhibits thrombin with an efficacy of ˜60%, whereas heparins have an efficacy of nearly 100% because their primary effector, antithrombin, is a covalent inhibitor of thrombin and other coagulation enzymes.1,34 This implies that even at saturation, the SbO4L-thrombin complex is likely to display a reduced level of pro-coagulant efficacy. This is a specific advantage of allosteric inhibitors and is the reason why these molecules are called regulators. SbO4L appears to possess this advantage and suggests its use as an effective prophylactic agent.
SbO4L allostery arises from binding in exosite 2 of thrombin, which also engages polymeric heparin. Although nearly ideal competition between heparin and SbO4L is indicated by competitive inhibition studies, it is not necessary that the two ligands are strictly mutually exclusive. Exosite 2 is a fairly large area spanning ˜20×30 Å2 and consisting of numerous electropositive residues including Arg93, Arg101, Arg165, Arg233, Lys235, Lys236, and Lys240. Of these, polymeric heparin recognizes Lys236, Lys240, Arg93, Arg101 and Arg233.43,44 Sulfated LMW lignins, the precursors of SbO4L, were found to bind to Arg93 and Arg175.17 Thus, the geometries of polymeric heparin and sulfated LMW lignins in exosite 2 of thrombin appear to be significantly different. Structurally, SbO4L is more heparin-like in terms of its sulfation density, but is more LMW lignin-like in terms of its hydrophobic scaffold. Thus, it is not clear whether SbO4L binding in exosite 2 will resemble heparin or sulfated LMW lignins. Yet, this geometry will determine whether SbO4L inhibits thrombin in a fibrin-bound state.
A key advantage in the use of polymeric heparins is the availability of protamine as an antidote. SbO4L is also a sulfated polymer and its direct inhibition of thrombin is neutralized by protamine. In fact in chromogenic test systems, protamine was found to restore 100% thrombin activity with a RC50 only 5-fold higher than IC50. Additionally, the effect was instantaneous as no extended incubation was necessary. An average UFH chain contains nearly 1.7 anionic groups (—OSO3− and —COO−) per monosaccharide, while SbO4L contains nearly two —OSO3− groups, which explains the highly effective protamine reversal.
Overall, the present work puts forward a novel designed sulfated β-O4 lignin molecule, SbO4L, as a highly selective and potent anticoagulant with considerable promise. SbO4L is exciting because it can be readily synthesized, is not derived from animals, is an allosteric regulator and its anticoagulation is likely to be reversed using the traditional heparin antidote, protamine.
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While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.
1. A SbO4L compound having a general chemical structure: where
- Y is SO3M or H, where at least one Y is SO3M;
- M is a positively charged ion;
- n ranges from 4-30;
- R1 is CH2OY; and
- R2, R3, R5, and R6 may be the same or different and are selected from the group consisting of hydrogen, C1-10 linear alkyl, C1-10 branched alkyl, C1-10 and O1-10 oxyalkyl, an electron-donating group, and electron-withdrawing group, a halogen, —NH2, and —NHR where R is selected from the group consisting of C1-10 alkyl, C1-10 aryl, C1-10 allyl, and benzyl,
- and wherein a sulfation density based on a difference in MW between the SbO4L compound and its unsulfated precursor is about 2 sulfate groups per monomer.
2. The SbO4L compound of claim 1, wherein at least a plurality of Y moieties are SO3M.
3. The SbO4L compound of claim 1, wherein M is independently selected from the group consisting of Na+, Ca2+, and NH4+.
4. The SbO4L compound of claim 1, wherein R1 is CH2OY in which Y is SO3M.
5. A sulfated low molecular weight lignin preparation comprising the SbO4L compound of claim 1 wherein n ranges from 4-6.
6. A sulfated low molecular weight lignin preparation comprising the SbO4L compound of claim 1 wherein n ranges from 5-10.
7. A sulfated low molecular weight lignin preparation comprising the SbO4L compound of claim 1 wherein n ranges from 10-25.
8. A sulfated low molecular weight lignin preparation comprising the SbO4L compound of claim 1 wherein n ranges from 15-30.
9. A mixture of two or more different sulfated lignin scaffolds, each of which is a SbO4L compound as set forth in claim 1.
10. A lignin containing composition comprising:
- one or more carriers or matrices; and
- one or more lignins, at least one of which is an SbO4L compound as set forth in claim 1.
11. The SbO4L compound of claim 1 wherein the general chemical structure is
12. The SbO4L compound of claim 11, wherein at least a plurality of Y moieties are SO3M.
13. A mixture of two or more different sulfated lignin scaffolds, each of which is a SbO4L compound as set forth in claim 11.
14. A lignin containing composition comprising:
- one or more carriers or matrices; and
- one or more lignins, at least one of which is an SbO4L compound as set forth in claim 11.
15. A method of making an SbO4L compound or mixture of SbO4L compounds as set forth in claim 1, comprising the steps of:
- polymerizing at least one monomer of a beta-O4 lignin compound to obtain a polymer;
- reducing the polymer produced in said polymerizing step; and
- sulfating the reduced polymers produced in said reducing step to a level of about 2 sulfate groups per monomer, compared to the reduced polymers.
16. The method of claim 15, wherein said at least one monomer is ethyl 2-bromo-3-(4-hydroxy-3-methoxyphenyl)-3-oxo-propanoate.
17. The method of claim 15 wherein said at least one monomer is only one monomer.
18. A method of making an SbO4L compound or mixture of SbO4L compounds as set forth in claim 11, comprising the steps of:
- polymerizing at least one monomer of a beta-O4 lignin compound to obtain a polymer;
- reducing the polymer produced in said polymerizing step; and
- sulfating the reduced polymers produced in said reducing step to a level of about 2 sulfate groups per monomer, compared to the reduced polymers.
19. A method of preventing or reducing coagulation of blood or plasma, comprising the step of adding to said blood or plasma an SbO4L compound as set forth in claim 1 or a mixture or composition containing said SbO4L compound to said blood or plasma.
20. The method of claim 19 wherein said step of adding is performed ex vivo.
21. The method of claim 19 wherein said step of adding is performed in vivo.
22. A method of preventing or reducing coagulation of blood or plasma, comprising the step of adding to said blood or plasma an SbO4L compound as set forth in claim 11 or a mixture or composition containing said SbO4L compound to said blood or plasma.
23. The method of claim 22 wherein said step of adding is performed ex vivo.
24. The method of claim 22 wherein said step of adding is performed in vivo.
Referenced Cited
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Foreign Patent Documents
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Patent History
Type: Grant
Filed: May 29, 2014
Date of Patent: Oct 3, 2017
Patent Publication Number: 20160115280
Assignee: Virginia Commonwealth University (Richmond, VA)
Inventors: Umesh R. Desai (Glen Allen, VA), Jay Thakkar (Denver, CO), Akul Mehta (Richmond, VA)
Primary Examiner: Nicholas Hill
Application Number: 14/889,870
International Classification: C07G 1/00 (20110101); C08H 7/00 (20110101); C07C 305/22 (20060101);


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Latest News on Pollinators
Showing of 0 - 10 from 3 results
Pollinators - Total results - 3
May 20, 2019
World Bee Day 2019: Honey, we shrunk the (number of) bees, we must bring them backWith a sharp decline in the number of bees, the food supply in India is under direct threat.
Feb 12, 2019
Collapse in world's plummeting insect numbers is threatening to destabilise natureInsects are going extinct at eight times the rate other animals are, which is worrying.
Dec 17, 2018
Scientists engineer bee vaccine to help the pollinator fight off 'insect apocalypse'Bees are vital pollinators in agriculture as they fertilise roughly 75% of food crops around the globe.

subset: commoncrawl


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Bottom Line: You won't obtain an improved overall on-line backup service than the full-highlighted IDrive, especially for the price.
Cloud storage is a snap to come back by. Dozens of services shovel a lot of totally free Place to you just for signing up. But which of All those services are thinking about the documents you add, and most importantly, which services encrypt your personal knowledge so no one can examine it? Let's Have a look.
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Because the appearance of the online market place, the engineering business has actually been steadily moving far from community storage to distant, server-based mostly storage and processing—what is recognized as the
Base Line: On the internet syncing and storage Device Box is user friendly and integrates with a wide array of apps and services, nonetheless it fees a whole lot a lot more than very similar products and solutions.
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Why waste important storage space on the Computer system or cellular phone if you can shop your documents and media in the cloud and share it throughout gadgets? These best-rated services let you do exactly that.
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Unveiling the field's very first neural network to protect significant infrastructure from cyber check here warfare
We're not saying it is best to drop your favorite service and transfer to among the types we stated, however, if you do keep in which you are, you should know what their privateness policy says. Dropbox, Google, and Amazon all say they are going to only dig into your documents if somebody strategies them off to unlawful or copyrighted product, or legislation enforcement will come knocking.
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This text may possibly consist of advertising, promotions, or affiliate one-way links. Subscribing to your e-newsletter suggests your consent to our Conditions of Use and Privacy Plan. It's possible you'll unsubscribe from your newsletters at any time.
The smart Trick of secure cloud service That No One is Discussing
Bottom Line: You won't obtain an improved overall on-line backup service than the full-highlighted IDrive, especially for the price.


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Guide to Get Your Girlfriend Rear – 5 Things You Need to Get Started
Will it be time to develop the man fall in love? Isn't it time to finally hear many three little words? Lots of people are wondering but if your fellow is on a single page because you? It'll probably want some operate on your behalf to build anyone fall in love. Suggestions some advice that may help you take him exact that you've currently reached.
Many times, women are selecting the body interior wrong way. They often make all sorts of things complicated by doing goods that aren't necessary – the makes all of them look unattractive. When you understand the easiest way to flirt using a man appropriately, things is going equally as you will planned it. To tell most people frankly, this really is just one difficulty indicating significantly. However you'll not always be flirting until you currently have interest in both instances. Only be careful while repeating this for yourself to not look too desperate. Here i will discuss the precise manner in employing your system parts when flirting.
Regarding the as the boomer period began or ended, it is not straightforward and is also also from subject controversy. For handy purposes, we shall have this to mean virtually anyone born around mid 1940s through around mid 1960s. It was also a period of great customizations in the entire world; socially, economically, politically and technically. foreign-brides official
Enhance the flavor you have to stop in accomplishing isn't really good mouthing people as part of your ex – partner's life who may have forked out to the breakup by his or her's actions or words. This is simply not productive whichever and you should look for another outlet for a distress feelings. If you can show that you may have grace pressurized, the individual would have much reverence on your behalf.
Step: Analyse your relationship. Ascertain what went wrong. Give attention to the negative (even if, especially presently, you should constantly forget everything that failed out). Be objective. Ask yourself if perhaps you actually want to build returning with her (not any, this is simply not a fabulous stupid question, even though that you're looking more knowledge about how for getting the woman's back! ). Many people sometimes turned out to be blind throughout a separation and know an unacceptable parts in the event that they're in the hazardous relationship.
3 Places Where You Can Easily Connect with Women and What to Do As you Get There
You need to attract a woman and get the girl's to enjoy intimacy on hand, then you most definitely ought to learn tips on how to touch her properly. Remember: if you want to grab a lady, you then must not try kissing her without having organized various laid back touching first. Here are some convenient techniques to build rapport without difficulty by making use of effect.
At this point, before we start making reference to the things they are curious about, shall we take your glance at what precisely they are not that fascinated by earliest. I found that unlike well-liked men's belief, what quantity of cash which you simply get may be a nonissue. So, by means of a good family car can be a and, it probably isn't a deal breaker in most of women of all ages. Critical Aspects For https://www.omicsonline.org/open-access/emphasize-on-niche-market-strategy-for-attracting-foreign-tourists-in-bangladesh-2162-6359-1000337.php?aid=73155 Across The Usa
When Eliezer prayed how the ideal girl would offer to consider virtually all his Camels water to help you drink, he was screening her to see if the girl was generous and favorable. Think of all the work this girl did serving those 15 Camels! Yet she made it happen willingly, for "she ran intended for the good to build additional liquid. " But King Louis Version lets us know the best way patients and careful Eliezer was once he seen:
Another method of check may be the modern-day look. This will be the classic appear you ought to check out a massive amount earlier men wearing. Some females like this search however, you should certainly consider in the event it trend is designed for you. With this look, you will generally keep the polo shirt or maybe a prolonged sleeve collar shirt through incorporating costume pants. If you attended a much more esteemed college, it's possible you'll decide the look as you will have the capability to match up with good.
some. Thank her for issues she's done. If the woman you happen to be complimenting will be the significant girl inside your lifetime, it is actually inevitable that she's performed several things for your needs out of the goodness of her heart. Maybe she's cooked you evening meal recently, or possibly she will be bought a brand-new shirt or shoes and boots. Allow her kudos to become just about the most clever girlfriend's you've ever had. Applaud her fantastic cooking talents to hire the girl's discover how substantially you appreciate all the items your lady does for your needs.


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@whtrbt Gluing the two planed faces together to make a good size leg.
@whtrbt scoring the line seems to help guide the saw?
Here is a long thread on keeping moss as a houseplant that I pulled off my twitter, wrapped up as a blog post - so now you can read about practical moss gardening without having to visit that website :>
Making aprons for the bench side. #theWorkshop
I made a photo blog on my site: https://phse.net/photo/
@whtrbt Smoothing out the bench top. Gotta decide whether to take off more to get rid of the gaps caused by the chamfered edge on the wood I laminated.
Making a workbench in my basement car park . Step 1... plane and laminate a heap of cheap pine. #theWorkshop
@whtrbt I did it! How nice has been meditating today, my back says thanks x1k!!!
For a long time I always preferred the half lotus because I couldn't stand the kneeled position for very long, but it's waaaay better for my back!
In the future I might want to make a slightly wider one, and also make it foldable like yours :)
@whtrbt oh, it's meant to be angled... I didn't mess up the cuts 😂
@whtrbt as I mentioned in my vipāssāna thread, I get pain in my knee... doesn't bother me much usually but staying in a fixed position (i.e. meditation) really exacerbates it. This really helps, but it's also a comfortable way to sit and work if you don't want to sit at a chair. And harder to slouch. :)
Just built myself a tiny bench so I can kneel for more than half an hour while meditating. :)
Used some Spotted Gum that was intended for decking, but it looks really nice. Added hinges so I can fold it up and also some felt padding on the feet.
Might make a small cloth bag to put it in that doubles as a thin cushion. :)
Revel in the marvels of the universe. We are a collective of forward-thinking individuals who strive to better ourselves and our surroundings through constant creation. We express ourselves through music, art, games, and writing. We also put great value in play. A warm welcome to any like-minded people who feel these ideals resonate with them. Check out our Patreon to see our donations.


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Psycho White dropping November 13th.
No Politics. No B.S.
Psycho White dropping November 13th.
Receive audio and video content exclusively to your inbox by signing up for the DEHH newsletter.
This was a surprise and came out of nowhere. At least to us it did. Listen: Juvenile-Juvie Tuesdays Related
Dee-1 and Mannie Fresh huh. Check out the song below to see what this sounded like. Related
Casual and J Rawls are dropping a joint project together, "Respect The Game Or Expect Flames", set for an August 28th release. First single below.


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North, South Korea Exchange Fire
North Korea has threatened military action against South Korea if the latter continues its "propaganda warfare," the South Korean news agency Yonhap reports. North Korea has previously called broadcasted messages near the border "a direct action of declaring a war." The two countries exchanged fire Thursday after North Korea fired at a speaker, with South Korea firing dozens of shells into its neighbor. Residents of South Korean border villages have been asked to relocate to shelters, in case the tense situation escalates.


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The Best Of Me
The Best Of Me is a TV show on Australian national television from SBS ONE with an average rating of 3.0 stars by TVCatchUpAustralia.com's visitors. We have 1 episodes of The Best Of Me in our archive. The first episode of The Best Of Me was broadcast in January, 2015. Did you miss an episode of The Best Of Me but don't you wan't that to happen in the future? Please set an alarm and add The Best Of Me to your favourites, so we can remind you by email when there's a new episode available to watch. Completely free: handy!
Episode of January 14, 2015
Expired 3.0 35 xA young woman's relationship is tested when she realises her boyfriend, who is in hospital with liver failure, has a double life she knows nothing about. Nominated for the Golden Leopard at the 2007 Locarno International Film Festival. Directed by Roser Aguilar and stars Marian Álvarez, Juan Sanz and Marieta Orozco. (In Sp...
Medicine Or Myth?Catch up on Medicine Or Myth? and watch online. Find the latest episodes of Medicine Or Myth?, broadcast by SBS ONE.
The World GameThe World Game is an Australian television show about football that covers reviews and previews of both national and international football and related issues.
24 Hours In Emergency24 Hours in Emergency is a British reality and medical television series that is set in the A&E department King's College Hospital in Denmark Hill (Camberwell) and later on at the A&E room at Saint George's Hospital in Tooting (Wandsworth).
French Open: Roland GarrosCatch up on French Open: Roland Garros and watch online. Find the latest episodes of French Open: Roland Garros, broadcast by SBS ONE.


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Advising Office
Schedule an appointment with a sociology staff advisor.
There are no regular drop-in hours during the summer. Please feel free to schedule an appointment with a staff advisor.
Sociology Peer Advisors will return in fall 2019.
Peer Advisors can:
-Help you clarify major requirements
-Tell you what it is like to be a Sociology or Organizational Studies major
-Help you plan your course schedule
-Share their own experiences about internships, undergraduate research, writing an honors thesis, studying abroad, going to UCDC or UCCS, double majoring, taking specific SOC courses, and more!
The Advising Office is closed from noon-1pm and available hours are subject to change.
For questions about General Education, Planned Educational Leave Program (PELP), or Permission to Drop (PTD), please consult the College of Letters and Science Advising Office.
Sociology Undergraduate Advisors
Caroline Herrod
Location: 1284 SS&H (please check in at 1282 SS&H)
Kelli Sholer
Location: 1285 SS&H (please check in at 1282 SS&H)
Mishaal Barrett
Location: 1287 SS&H (please check in at 1282 SS&H)


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So you have been diagnosed with Diabetes Mellitus? You may feel devastated at this very moment but certainly, feeling depressed and hopeless won't do you any good. Getting to know more about this certain disease, its types, treatment methods, and the like is very important for a diabetic's healthy and normal functioning.
Diabetes Mellitus occurs when a person's insulin level is absent or deficient. Insulin serves as the key for the glucose in our blood to enter the cellular environment where it will be used to power up the cells. With a deficiency or absence of insulin, glucose cannot enter the cells and therefore cannot be metabolized. The amount of glucose in our blood accumulates leading to a high level of blood sugar.
Basically, there are two types of Diabetes Mellitus—Type I or Insulin Dependent and Type II or Non Insulin Dependent. Type I Diabetes Mellitus is caused by the predominant inability of the pancreas to produce insulin—which means there is an absolute absence of insulin. Type II Diabetes Mellitus is called Non-insulin dependent DM in which the production of insulin is deficient, but there is the presence of insulin in the blood.
Each type of Diabetes Mellitus has a specific complication—both of them are fatal. For Type I DM, DKA or Diabetic Ketoacidosis occurs. As for Type II DM, the fatal complication that may arise is called HHNK Syndrome or Hyperosmolar Hyperglycemic Non-Ketotic Syndrome, orsimply Hyperosmolar Hyperglycemia.
How Hyperosmolar Hyperglycemia Occurs?
Hyperosmolar hyperglycemia occurs with three cardinal situations—an extremely high blood sugar level, extreme dehydration or lack of body fluids and a low level of consciousness.
In the normal physiology, our kidneys are organs which compensate in case our body has a high amount of glucose. It does its job by allowing an amount of glucose to exit our body through our urine. However, without proper hydration and continuous intake of sugary foods, our kidneys won't be able to get rid of these excess sugars. As a result, our blood sugar levels will further shoot up resulting to a high concentration of blood—giving rise to hyperosmolarity. When our blood becomes hyperosmolar, it has the tendency to rob the fluids out of body organs leading to dehydration of body organs.
Symptoms of Hyperosmolar Hyperglycemia
Confusion and lethargy may happen at an early onset, accompanied with increased urination and increases thirst. Ultimately, coma may happen in a patient with Hyperosmolar Hyperglycemia.
Treatment of Hyperosmolar Hyperglycemia
The main goal of treatment for Hyperosmolar Hyperglycemia is correcting the dehydration. Intravenous fluids as well as potassium chloride may be administered to correct and improve the condition of the body's hydration status.
Hyperosmolar Hyperglycemia is a medical emergency which means once any of these symptoms present, be sure to contact emergency services for a more immediate form of treatment. Realizing the hyperosmolar hyperglycemia symptoms is very important to avoid complications which may arise such as circulatory failure, acidosis and cerebral edema… and ultimately death. Most of all, the key to preventing Hyperosmolar Hyperglycemia is to properly manage Diabetes Mellitus.


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18 Comments
Is that a drug head talking. Please. Listen to that lady. Ignorance. Herb is not addictive. Rum and cigarettes kill the most people every year. Check the stats. Pure emotional rubbish that white guy talking. People smoke herb in my country like how you eat mangoes and i dont see those things happening. You can get addictive to anything. Sex money work parties sleeping. As the lady said moderation.
Herb was from the biginning. Because of your man made laws the world is turned upside down. Madness. And herb is a plant not a drug.
Correlation does not imply causation. It's the number one rule of gathering and applying data. There is a correlation between Marijuana use and other drug use. This does NOT mean the one led to the other. Most people who use dagga do not become dependent or exhibit other drug seeking behaviors. Most Drug addicts have will have abused other substances like alcohol. You have as much proof that drinking water causes people to become drug addicts as you do to say Dagga caused it. It's like saying drinking water causes death because 100% of all people who have ever died have also drunk water. Yes, there is a correlation between drinking water and death but the relationship is not causal.
This Rasta speaks well but makes No Sense at all – He is not the one people come to when they have problems because of using cannabis.
There is no Scientific evidence that the use of Marijuana leads to any psychological effects. These three people are spewing populist rhetoric and have no evidence or facts to backup their claims. The rasta lawyer has been smoking it probably for YEARS, is he showing any kind of Psychological effects?? If they did their research properly, they'd know that cannabis has two main compounds, TCH (tetrahydrocannabinol ) and CBD (cannabidiol). TCH is the component that causes the feeling of "Euphoria", whereas CBD reverses that feeling. Both compounds have proven to have much medical benefits to fight various diseases. See https://www.healthline.com/health/medical-marijuana
The only reason why there was ever the big push against Marijuana, is because this drug can replace most useless medicines manufactured by Big Pharma in the cure for various cancers and other fatal diseases.
First of all people including some cannabis advocates must stop referring to cannabis as a "DRUG". It is a plant that has been around for thousands of years and probably the first cultivated crop. Furthermore it is natural unlike drugs such as coke ,heroin etc.whose original plants etc have been altered using toxic chemicals etc.Addiction is a choice. Nobody from my research has ever died from the direct ingestion of this much maligned plant. Alcohol and its abuse on the other hand has caused direct death . This of course includes many other drugs. Non steroidal antiinflametories on the other hand cause massive rates of fatalities across the world. Cannabis has been demonised by various nations and institutions for decades to further their own agendas. Mostly for financial gain and political control over local populations that they colonised. To give an example England banned and demonised Hemp Sativa in Egypt so it would not impact on their cotton business to mention only one of many examples. Racism and subjugation was also part of the plan there and many countries across the globe.Now big business wants to get involved together with government and medical control council etc once again to profit from one of natures gifts. So they now realise its potential to heal as well as aliviate many health issues They take a plant that has hundreds of canabanoid and terpenes and pick on one compound CBD and try and sell that concept to the public and other ill informed people and make it socially acceptable. Once again making millions for their shareholders etc. Don't buy into that lie. The effect of cannabis for medical use is synergistic and all the compounds need to work together. Not saying CBD is bad but its not the whole story. That person who claims to have had personal experience with addiction etc is ill informed and biased. Cannabis is not a Gateway Drug. I myself have had a family member addicted to tik and other drugs and he never touched "DAGGA". He made a choice to take drugs for whatever reason. He also made a choice to get off drugs and stay clean.So just to wrap up i don't drink heavily and i don't smoke cannabis although i did in my youth. We did drink excessively and party like any young person that has found their new freedom. Some of my peers did ruin their lives and became alcoholics etc. Again a choice. By the way most people that smoke or vape weed do not even smoke cigarettes. They also do not get Paralytic drunk beat up their wife and kids kill people on our roads fight with neighbors etc. Don't get me wrong i am all for braaing and watching rugby etc and having a few beers. I just don't drive in that case. STOP blaming weed for all the ills of society. And don't buy into the false propaganda no matter what Experts that they have on their payroll trying to convince you.
Wake the hell up people. The p[lant was banned becuase it intefered with profits from other organisations. Fact. Textile, Medicinal, Tobacco, Petrolchemical, etc etc etc. Its been proven to be beneficial to human health.
A very good arguments by Adam, was very informative, thanks
I cannot believe we are letting drug addicts make the rules now… how irresponsible SA, but I guess now no one has to convince anyone that dagga harms anymore… those in doubt will see it for themselves unfold… and those who are deceiving the rest will be exposed. These people want to make money off the suffering and addiction of SA citizens.
Everybody is making a noise about the ruling. Nothing is going to change. You are not going to get more people smoking. You are now going to realise how many people actually smoke dope. The difference will be that they don't have to fear arrest.
The guy on TV is sick, Prince has been on this for ages his research is outstanding he's a lawyer after all.
I have never smoked weed and said you know what will be dope….shooting some heroine in my vain.
white man needs to stop with Lsd and smoke weed. no facts just assumptions
Is it just me or is the host bias? Don't even bother paying mind to the white guy.
This Rusta knows what he is talking about as he has been doing this research for years. You can't call weed a drug its no longer a drug. Adam has no idea what he is talking about. Just because he was addicted to other drugs does not mean that all what he went through is the same. Adam get over yourself please come on. Weed does not kill. ALSO IT WAS STATED THAT ADULTS ONLY. so kids can not take it. So parents should be more parents to their kids. I cannot believe you would bring someone in who has no clue what he is on about (Adam). The USA and other countries have been doing research on this for so many years and there is also HUGE health benefits to use it. One can not be so uneducated to think that all the work the Rasta would ask for this to be put through and have no evidence to prove his case. How can we blame drugs, alcohol etc as the problem. It's not educating the youth and adults of this country. And there is not enough help for other problems and now you want to say Adam that you were in the same thing. No you uded harmful drugs that caused issues to your health. You can not speak on behalf of all people. As smoking tabaco and alcohol, anorexia, overweight etc causes health and mental health issues. I do not smoke weed. But my husband has epilepsy and canibas could help with that. Just like very thing can be addictive. Porn, gambling, playing games, sex etc. We can not as a country grow and have freedom if we cannot come together and help with educating everyone. Yes there will be other laws with weed, but that will help for our future. Please can you do your homework before bring people onto your show that have no cooking clue what the topic is about. The Rusta (thumbs up) I behind you 100%
This panel is ridiculous. Everything they said about marijuana is true for alcohol and tobacco. Yet they are fine with those not being criminalised.
Criminalisation won't solve addiction. It just forces people to go to the black market for their fix.
Why does ENCA get the propaganda machinery crew
this whit man is crazy , no facts at all


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Here's my calendar if you want to get together
On March 17, 2019 I added this calendar. The calendar idea is just that, an idea of where I may be in the city of Boulder, Colorado (Google Maps). The concept is to be accountable, transparent and accessible. Another thought is that since my ministry is public, I may publicly show my ministry events and actions. This could also be for my Patrons who support this missionary ministry. At least, this is what I'm going to attempt… you can also contact me here. We'll see how this goes! —JC
Personal Work Day - Tuesday Trading!
May 28, 2019 –
This is something new for me. I think the Lord is allowing me to trade on Tuesday's for the entire trading day: 7:30 am - 2:00 pm. I'll let you know next week how it went.
Teach me Lord to number my days in wisdom
So teach us to number our days, that we may apply our hearts unto wisdom. – in Psalm 90:12 , KJV