Chaperone therapy update: Fabry disease,GM1-gangliosidosis and Gaucher disease

Chaperone therapy is a newly developed molecular therapeutic approach to lysosomal diseases, a group of human genetic diseases causing severe brain damage. Based on early molecular studies during the last decade of the 20th century and early years of the 21st century, mainly on Fabry disease and G_M1-gangliosidosis, we found some mutant enzyme proteins were unstable in the cell, and unable to express catalytic activities. Subsequently galactose and other active-site binding substrate analogs were found stabilized and enhance the mutant enzyme activity in culture cells. We concluded that the mutant misfolding enzyme protein and substrate analog competitive inhibitor (chemical chaperone) form a stable complex to be transported to the lysosome, to restore the catalytic activity of mutant enzyme after spontaneous dissociation under the acidic condition. This gene mutation-specific molecular interaction is a paradoxical phenomenon that an enzyme inhibitor in vitro serves as an enzyme stabilizer in situ. First we developed a commercially available compound 1-deoxygalactonojirimycin (DGJ) for Fabry disease, and confirmed the above molecular phenomenon. Currently DGJ has become a new candidate of oral medicine for Fabry disease, generalized vasculopathy involving the kidneys, heart and central nervous system in the middle age. This drug development has reached the phase 3 of human clinical study. Then we found two valienamine derivatives, N-octyl-4-epi-β-valienamine (NOEV) and N-octyl-β-valienamine (NOV), as promising therapeutic agents for human β-galactosidase deficiency disorders (G_M1-gangliosidosis and Morquio B disease) and β-glucosidase deficiency disorders (phenotypic variations of Gaucher disease), respectively. Originally NOEV and NOV had been discovered as competitive inhibitors, and then their paradoxical bioactivities as chaperones were confirmed in cultured fibroblasts from patients with these disorders. Subsequently G_M1-gangliosidosis model mice have been used for confirmation of clinical effectiveness, adverse effects and pharmacokinetic studies. Orally administered NOEV entered the brain through the blood–brain barrier, enhanced β-galactosidase activity, reduced substrate storage, and improved neurological deterioration clinically. Computational analysis revealed pH-dependent enzyme-chaperone interactions. Our recent study indicated chaperone activity of a new DGJ derivative, MTD118, for β-galactosidase complementary to NOEV. NOV also showed the chaperone effect toward several β-glucosidase gene mutants in Gaucher disease. Furthermore a commercial expectorant drug ambroxol was found to be a chaperone for β-glucosidase. A few Gaucher patients responded to this drug with remarkable improvement of oculomotor dysfunction and myoclonus. We hope chaperone therapy will become available for some patients with Fabry disease, G_M1-gangliosidosis, Gaucher disease, and other lysosomal storage diseases particularly with central nervous system involvement.     

Genetically engineered mouse models of the trinucleotide-repeat spinocerebellar ataxias

The spinocerebellar ataxias (SCAs) are dominantly inherited disorders that primarily affect coordination of motor function but also frequently involve other brain functions. The models described in this review address mechanisms of trinucleotide-repeat expansions, particularly those relating to polyglutamine expression in the mutant proteins. Modeling chronic late-onset human ataxias in mice is difficult because of their short life-span. While this potential hindrance has been partially overcome by using over-expression of the mutant gene, and/or worsening of the mutation by increasing the length of the trinucleotide repeat expansion, interpretation of results from such models and extrapolation to the human condition should be cautious. Nevertheless, genetically engineered murine models of these diseases have enhanced our understanding of the pathogenesis of many of these conditions. A common theme in many of the polyglutamine-repeat diseases is nuclear localization of mutant protein, with resultant effects on gene regulation. Conditional mutant models and transgenic knock-down therapy have demonstrated the potential for reversibility of disease when production of mutant protein is halted. Several other genetically engineered murine models of SCA also have begun to show utility in the identification and assessment of more classical drug-based therapeutic modalities.     

CD39/NTPDase-1 activity and expression in normal leukocytes

INTRODUCTION: CD39/NTPDase-1 is a cell surface enzyme expressed on leukocytes and endothelial cells that metabolizes ATP to ADP and AMP. CD39 is expressed on numerous different types of normal leukocytes, but details of its expression have not been determined previously. METHODS: We examined CD39 expression and activity in leukocytes isolated from healthy volunteers. Expression of CD39 on leukocytes was measured by FACS and activity of CD39 in lymphocytes and neutrophils was determined by an enzymatic radio-TLC assay. RESULTS: We established that CD39 is expressed on neutrophils, lymphocytes, and monocytes. The enzyme is found on >90% of monocytes, neutrophils, and B-lymphocytes, and 6% of T-lymphocytes and natural killer cells. Per cell density of expression varied, with the highest expression on monocytes and B-lymphocytes. ATPase and ADPase activities were highest on B-lymphocytes, lower on neutrophils, lowest on T-lymphocytes. The ratio of ADPase:ATPase activity was 1.8 for neutrophils and B-lymphocytes and 1.4 for T-lymphocytes. Hypertensive volunteers had lower levels of CD39 on their T-lymphocytes and NK cells. No correlation between age, gender, ethnic background, or cholesterol level and CD39 expression was observed. CONCLUSIONS: We conclude that CD39 activity and expression are present to varying degrees on all leukocytes types examined. Differences between leukocyte types should be considered when examining CD39 in disease states.     

The ratio of ADP- to ATP-ectonucleotidase activity is reduced in patients with coronary artery disease

INTRODUCTION: CD39 (NTPDase1), an endothelial cell membrane glycoprotein, is the predominant ATP diphosphohydrolase (ATPDase) in vascular endothelium. It hydrolyses both triphosphonucleosides and diphosphonucleosides at comparable rates, thus terminating platelet aggregation and recruitment responses to ADP and other platelet agonists. This occurs even when nitric oxide (NO) formation and prostacyclin production are inhibited. Thus, CD39 represents the main control system for platelet reactivity. Reduced or deficient local ecto-nucleotidase activity may predispose to development of vascular disease. Based on data in animal models and in vitro, CD39 constitutes a new therapeutic modality for vascular disease with a novel and unique mode of action. MATERIALS AND METHODS: Lymphocytes were isolated from 46 patients with angiographically proven coronary artery disease (CAD) as well as from matched healthy control subjects. Ectonucleotidase ADPase and ATPase activities (prototypical for the ATPDase activity of endothelial cells) were measured using established radio-TLC procedures. RESULTS AND DISCUSSION: In the patients, a decreased ratio of ADPase to ATPase activities (from 1.26 to 1.04) was observed despite increases in both ADPase and ATPase activities. Coronary artery disease was the only independent predictor of a difference in the ADPase/ATPase activity ratio by multivariate linear regression analysis (P=0.0035). This altered ADPase/ATPase activity ratio in patients may represent a reduction in endogenous defense systems against platelet-driven thrombotic events. These data may identify a population of patients with excessive platelet reactivity in their circulation. Increased generation of prothrombotic ADP in these patients implies a potential benefit from therapeutic intervention with soluble forms of CD39.     

Determination of platelet factor 3 in whole blood by a chromogenic peptide substrate assay

A newly developed chromogenic peptide substrate assay for the determination of platelet factor 3 (PF 3) in plasma was modified for application to whole blood. The PF 3 activity values found in whole blood from healthy volunteers were essentially the same as those of the corresponding platelet-rich plasma samples. The PF 3 values obtained in whole blood taken in the anti-coagulant EDTA/citrate/PGE 1/ /theophylline were stable for three hours or more after drawing the blood, which indicated that no platelet release had taken place. When citrate was used as an anticoagulant a marked increase in PF 3 activity started 10 to 20 minutes after drawing the blood. Release of β-thromboglobulin (β-tg) was found to occur simultaneously with the release of PF 3 under these conditions. This means that normal transfusion blood contains a large amount of PF 3 activity as well as other platelet release products. Infusion of such material into patients subjected to major surgery or trauma might increase the risk of thrombosis or disseminated intravascular coagulation. Generation of PF 3 activity in platelet-rich plasma upon addition of collagen and during blood clotting has been studied.     

Prediction of the therapeutic index of marketed anti-coagulants and anti-platelet agents by guinea pig models of thrombosis and hemostasis

Introduction

Animal models of thrombosis and hemostasis are critical for target validation in pharmaceutical research. Guinea pig haemostatic mechanisms, such as the platelet thrombin receptor repertoire, resemble those of humans. Measuring the performance characteristics of marketed antithrombotic drugs in guinea pig models is a key to predicting therapeutic indices of new agents. The goal of the current study was to benchmark representative marketed drugs in thrombosis and hemostasis models in guinea pigs.

Methods

Effects of the cyclooxygenase inhibitor, aspirin, the P2Y₁₂ antagonist, clopidogrel, the glycoprotein IIb/IIIa inhibitor, tirofiban, and the direct thrombin inhibitors, argatroban and hirudin, were evaluated in this study. Antithrombotic agents were tested in FeCl₃-induced carotid artery thrombosis and arterio-venous shunt thrombosis models. Haemostatic effects of drugs were evaluated in cuticle and renal bleeding models. Ex vivo measurements of platelet function and coagulation inhibition were performed to benchmark preclinical doses of each agent to those used clinically.

Results

The overall rank-order of potency in thrombosis models based on per cent of vessels occluded, average carotid blood flow, and thrombus weight was aspirin = argatroban = tirofiban < hirudin = clopidogrel. In bleeding models, the rank order was: aspirin < clopidogrel = argatroban = tirofiban < hirudin.

Conclusion

This characterization of representative drugs from two important classes of anti-coagulant and anti-platelet agents in efficacy and bleeding models in guinea pigs provides a reference point for evaluation of new antithrombotic agents.     

Experimental study of biosoluble drugs. Thrombus lysis with biosoluble immobilized fibrinolysin in experiment.

In the animal experiments the strong thrombolytic effect of fibrinolysin immobilized on modified Sephadex was shown. In all cases after intraarterial administration of the preparation obtained the recovery of the blood flow in the femoral artery and the normal angiographic picture took place. There was no recovery of practicability in the control artery without immobilized fibrinolysin administration after the artificial thrombosis. The use of thrombolytic enzymes in suggested modification has number of advantages: the carrier with the enzyme is fixed in the vicinity of the thrombus and the dose of the enzyme can be by several orders lower than usual therapeutic dose.     

Cleavage of fibrinogen by human platelet calcium-activated protease

In lysates of washed human platelets produced by sonication or by addition of nonionic detergent, fibrinogen (Mr 340,000) was rapidly degraded, under conditions favorable to activation of the endogenous calcium-activated protease (CAP), to a core derivative (Mr 280-290,000) composed of partially degraded A alpha chains (Mr 47,000, 46,000, and 34,000) and B beta chains (Mr 56,000), and apparently intact gamma chains (Mr 53-54,000). Extensive degradation occurred within one minute at 4 degrees C, ambient temperature or at 37 degrees C, and was inhibited by leupeptin, EDTA, EGTA, or N-Ethylmaleimide, but not by soybean trypsin inhibitor, hirudin, aprotonin, benzamidine, phenylmethylsulfonyl fluoride or epsilon-aminocaproic acid. Purified plasma fibrinogen exposed to lysates containing active protease was cleaved in an identical fashion. The cleavage pattern of A alpha chains produced by this platelet protease activity is different from that produced by plasmin in vitro or that found in fibrinogen catabolites in vivo, and is unlike that produced by any cellular fibrinolytic enzyme yet described. In view of this finding, as well as the striking differential inhibitory effect of the agents cited above, we conclude that the degradation of platelet fibrinogen observed in these studies is due to direct proteolysis by platelet CAP.     

The Kallikrein Inhibitor from Bauhinia bauhinioides (BbKI) shows antithrombotic properties in venous and arterial thrombosis models

The Bauhinia bauhinioides Kallikrein Inhibitor (BbKI) is a Kunitz-type serine peptidase inhibitor of plant origin that has been shown to impair the viability of some tumor cells and to feature a potent inhibitory activity against human and rat plasma kallikrein (K_iapp 2.4 nmol/L and 5.2 nmol/L, respectively). This inhibitory activity is possibly responsible for an effect on hemostasis by prolonging activated partial thromboplastin time (aPTT). Because the association between cancer and thrombosis is well established, we evaluated the possible antithrombotic activity of this protein in venous and arterial thrombosis models. Vein thrombosis was studied in the vena cava ligature model in Wistar rats, and arterial thrombosis in the photochemical induced endothelium lesion model in the carotid artery of C57 black 6 mice. BbKI at a concentration of 2.0 mg/kg reduced the venous thrombus weight by 65% in treated rats in comparison to rats in the control group. The inhibitor prolonged the time for total artery occlusion in the carotid artery model mice indicating that this potent plasma kallikrein inhibitor prevented thrombosis.     

Antithrombotic and anticoagulant effects of wild type and Gla-domain mutated human activated protein C in rats

The antithrombotic and anticoagulant effects of recombinant wild type (WT) and mutated human activated protein C (hAPC) were investigated using a rat model of arterial thrombosis. Recent in vitro studies using human plasma have shown enhanced anticoagulant effects of hAPC by mutagenesis of either loop 148 in the serine protease domain or of the Gla domain. The Gla-domain mutant QGNSEDY-hAPC (= H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y) was found to be particularly active as an anticoagulant. We now combined the two mutations to create the variant QGNSEDY-hAPC:B148 and investigated the in vivo effects of this variant as well as of QGNSEDY-hAPC and WT hAPC using a rat model of arterial thrombosis. In vitro clotting experiments using rat plasma demonstrated WT hAPC to be inefficient, whereas both mutant hAPC variants yielded distinct dose dependent anticoagulant effects. In the arterial injury model, a segment of the left common carotid artery was opened longitudinally. An endarterectomy was performed and the arteriotomy was closed, whereafter the vessel was reperfused and the patency rate determined after 31 min. Three treatment groups each containing 10 rats and a control group of 20 animals were in a blind random fashion given intravenous bolus injections of 0.8 mg/kg WT or mutant hAPC or vehicle only. The ex vivo clotting times of plasma drawn 3 min after the injections, as compared to baseline clotting times, were approximately doubled by QGNSEDY-hAPC and tripled by QGNSEDY-hAPC:B148 infusions, while WT APC had little effect. Compared to the control group, none of the hAPC preparations had significant antithrombotic effect or increased arteriotomy bleeding.     

Purification and partial characterization of adenosine diphosphatase activity in bovine aorta microsomes

Anti-aggregatory activities in bovine aorta microsomal fractions were solubilized with Triton X-100 and separated into two fractions by DEAE-Sepharose CL-6B. One fraction strongly inhibited arachidonic acid-induced platelet aggregation, and the other inhibited ADP-induced aggregation. The latter fraction contained ADPase activity. The ADPase activity was further purified by affinity chromatography. The purified enzyme had specific activities of 43.8 and 48.2 mumol of Pi/min/mg protein for ADP and ATP, respectively. The enzyme required calcium or magnesium ions and it was insensitive to ATPase inhibitors, namely oligomycin and ouabain, and to adenylate kinase inhibitor, Ap5A. Polyacrylamide gel electrophoretic experiments indicated that only one enzyme was involved. This was confirmed by the parallel behavior of ADPase and ATPase activities throughout all the purification steps. These results suggest that the main anti-aggregatory activity of bovine aorta microsomes for ADP-induced aggregation is due to an ATP diphosphohydrolase (EC 3.6.1.5).     

Purification, biochemical properties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model

Introduction

The prevalence of cardiovascular diseases, one of the major causes of worldwide mortality, is being increasingly reported. Safer, more effective, and less expensive thrombolytic drugs can possibly overcome the underlying problems associate with current thrombolytic drugs.

Methods

A thrombolytic enzyme was purified and characterized from a Streptomyces strain. Carrageenan induced tail-thrombosis mice model was used to evaluate in vivo antithrombotic effect of the enzyme.

Results

First 15 N-terminal amino acids of the purified enzyme were IAGGQAIYAGGGRRS, which are significantly different from the reported fibrinolytic enzymes. The enzyme exhibited 14.3 ± 2.3-fold stronger thrombolytic activity than that of plasmin. In carrageenan induced tail-thrombosis model, the enzyme caused reduction in frequency of thrombus. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose.

Conclusions

The enzyme purified from the Streptomyces strain can be a potential candidate for the treatment of thrombosis.     

Effects of antidepressants on mRNA levels of antioxidant enzymes in human monocytic U-937 cells

Alterations of antioxidant enzyme activities have been described in a number of psychiatric disorders including major depression. Subsequently, the present study examined the effects of different types of antidepressants (desipramine, imipramine, maprotiline and mirtazapine) in different concentrations (10(-5), 10(-6) and 10(-7) M) on the mRNA levels of various enzymes of the antioxidant system, including both intracellular superoxide dismutase isoforms, glutathione peroxidase and catalase as well as several enzymes of the glutathione metabolism in monocytic U-937 cells after short- and long-term treatment (2.5 and 24 h) via RT-PCR. Results indicated mainly short-term decreases in the mRNA levels of antioxidant enzymes after treatment with these substances in all the concentrations used. In addition, after long-term treatment, significant increases in the mRNA levels were seen in the cases of Cu, Zn superoxide dismutase, gamma-glutamyl-cysteine synthetase, glutathione-S-transferase and glutathione reductase, including the impacts of all the antidepressants used in concentrations of 10(-6) M and 10(-7) M. Based on the large number of significant effects of all types of antidepressants tested on various antioxidant enzymes, we suggest that antioxidant enzymes may represent important targets in the course of antidepressive treatment.     

Amyloid-degrading enzymes as therapeutic targets in Alzheimer's disease

The steady state concentration of the Alzheimer's amyloid-beta peptide in the brain represents a balance between its biosynthesis from the transmembrane amyloid precursor protein (APP), its oligomerisation into neurotoxic and stable species and its degradation by a variety of amyloid-degrading enzymes, principally metallopeptidases. These include, among others, neprilysin (NEP) and its homologue endothelin-converting enzyme (ECE), insulysin (IDE), angiotensin-converting enzyme (ACE) and matrix metalloproteinase-9 (MMP-9). In addition, the serine proteinase, plasmin, may participate in extracellular metabolism of the amyloid peptide under regulation of the plasminogen-activator inhibitor. These various amyloid-degrading enzymes have distinct subcellular localizations, and differential responses to aging, oxidative stress and pharmacological agents and their upregulation may provide a novel and viable therapeutic strategy for prevention and treatment of Alzheimer's disease. Potential approaches to manipulate expression levels of the key amyloid-degrading enzymes are highlighted.     

Inhibition of human platelet aggregation by vitamin K

The effect of several vitamin K (Vit K) analogues on the aggregation of human platelets was examined. The analogues were potent inhibitors of aggregation induced by ADP, thrombin, collagen and arachidonate but were less active against aggregation induced by the calcium ionophore A23187. Vit K3 also prevented platelet membrane phosphatide breakdown induced by collagen. These effects were not due to a direct inhibition of enzymes involved in the liberation of arachidonate or its subsequent transformation. The analogues exerted no effects on enzymes regulating intraplatelet cAMP. However, these effects could be overcome by increasing extracellular Ca++ levels, indicating a possible interaction with Ca++ regulation in platelets.     

Simplified recombinant plasmin: production and functional comparison of a novel thrombolytic molecule with plasma-derived plasmin

A simplified and fully functional deletion mutant of plasminogen was created in which the middle portion of the molecule was removed, resulting in kringle 1 attachment to the serine protease domain. This recombinant plasminogen deletion mutant, Delta(K2-K5)Pg, was produced in the form of inclusion bodies at the yield of up to 200 mg/l in an Escherichia coli T7 expression system. Following protein refolding and purification on lysine-Sepharose, the conversion of the recombinant molecule Delta(K2-K5)Pg to the active enzyme mutant Delta(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied. Properties of Delta(K2-K5)Pg were similar to native, human plasma-derived plasminogen. Delta(K2-K5)Pg effectively bound epsilon-aminocaproic acid (K(d) = 11.3 +/- 2.3 muM) and fibrin (C(50) approximately 0.3 muM). The plasminogen activators streptokinase, urokinase, and tissue plasminogen activator effectively converted the recombinant zymogen Delta(K2-K5)Pg to the active recombinant enzyme, Delta(K2-K5)Pm. Additionally, Delta[K2-K5]Pm was rapidly inhibited by alpha(2)-antiplasmin (1.1 +/- 0.1 x 10(7) M(-1)s(-1)) and alpha(2)-macroglobulin (7.6 +/- 0.6 x 10(5) M(-1)s(-1)). In an in-vitro model, Delta(K2-K5)Pm demonstrated fibrinolytic potency comparable to human plasma-derived plasmin. Because of their unique biochemistry, including fibrin-binding properties and rapid inhibition by alpha(2)-antiplasmin, both native plasmin and a simplified deletion mutant of plasmin are potentially safe and effective direct thrombolytic agents for various thrombotic conditions. Further studies evaluating the in-vivo pharmacologic safety and clinical efficacy of this simplified plasmin (i.e. Delta[K2-K5]Pm) are warranted.     

Four missense mutations identified in the protein S gene of thrombosis patients with protein S deficiency: effects on secretion and anticoagulant activity of protein S

Four missense mutations, G54R, T589I, K155E, and Y595C, were identified in the protein S (PS) gene of the patients with PS deficiency and venous thrombosis. Three patients were heterozygous for the novel mutations, G54R, T589I, and Y595C, while a remaining one patient was homozygous for the K155E mutation, which is known to be a polymorphism in the Japanese population. A family study revealed that the Y595C mutation was associated with a Type I PS deficiency and the K155E mutation with a Type II PS deficiency, while no family study was performed for the patients with the G54R and T589I mutations. To determine whether these four mutations play a causative role in PS deficiency, the four PS mutants and wild-type PS were stably expressed in human embryo kidney (HEK) 293 cells. Pulse-chase experiments showed intracellular degradation and decreased secretion of the Y595C mutant. In the activated protein C (APC) cofactor assays, the specific activity of the K155E mutant decreased to 58% of that of wild-type PS. The APC cofactor activity of the three mutants, G54R, K155E, and T589I, were inhibited by C4b-binding protein (C4BP) with a dose dependency similar to that of wild-type PS. These results indicate that the Y595C and the K155E mutations are responsible for a secretion defect and a decreased anticoagulant activity of PS, respectively. The remaining two mutations, G54R and T589I, however, did not produce any definite abnormality leading to a low plasma PS activity.     

An optimized murine model of ferric chloride-induced arterial thrombosis for thrombosis research

INTRODUCTION/OBJECTIVES: Animal models are important tools in thrombosis research and preclinical drug development. Ferric chloride has been widely used to induce arterial thrombosis in a variety of species. However, almost all previous reports applied a very high concentration of ferric chloride (10-60%) to induce thrombus formation leading to difficulties in evaluating the efficacy of antithrombotic agents. Thus, the purpose of this study was to refine the ferric-chloride-induced thrombosis model to be better suited for thrombosis research. METHODS AND RESULTS: Dose-dependent study was used to identify a threshold concentration of ferric chloride sufficient for consistent occlusion (as reflected by the Doppler blood flow) of the carotid artery in C57BL/6 mice. Ferric chloride concentration at or about a threshold of 2.5% was found to be sensitive to anticoagulant (e.g., heparin) and antiplatelet (e.g., clopidogrel) agents. In contrast, the vessel rapidly occluded at or above 5% ferric chloride concentration despite pretreatment with the antithrombotic agents, even at doses that exerted maximal prolongation of tail bleeding time. CONCLUSIONS: Our study provides a simple, sensitive and highly controlled method for limiting vessel injury in mice to better detect the efficacy of antithrombotic drugs and/or evaluate therapeutic targets.     

Inhibition of platelet aggregation by curdione from Curcuma wenyujin essential Oil

Introduction

Curdione, one of the major sesquiterpene compounds from Rhizoma Curcumae, has been shown to exhibit multiple bioactive properties. In this study, we investigated the anti-platelet aggregation and antithrombotic activities of curdione with different methods both in vitro and in vivo. The purpose of the study was to explore an inhibitor of platelet aggregation, which promised to be a preventive or therapeutic agent for various vascular diseases.

Materials and Methods

Curdione was isolated from the essential oil of Curcuma wenyujin using the silica gel column chromatography method. The effects of curdione on human platelet aggregation induced by thrombin (0.3 U/ml), platelet-activating factor (PAF, 0.375 μg/ml), adenosine diphosphate (ADP, 10 μM) and arachidonic acid (AA, 0.1 mg/ml) were tested in vitro, and the potential mechanisms underlying such activities were investigated. We also tested the antithrombotic effect of curdione in a tail thrombosis model.

Results and conclusions

Curdione preferentially inhibited PAF- and thrombin- induced platelet aggregation in a concentration-dependent manner (IC₅₀: 60-80 μM), whereas much higher concentrations of curdione were required to inhibit platelet aggregation induced by ADP and AA. Curdione also inhibited P-selectin expression in PAF-activated platelets. Moreover, curdione caused an increase in cAMP levels and attenuated intracellular Ca²⁺ mobilization in PAF-activated platelets. In vivo, we also found that curdione showed significant antithrombotic activity. Therefore, we conclude that the inhibitory mechanism of curdione on platelet aggregation may increase cAMP levels and subsequently inhibit intracellular Ca²⁺ mobilization. Furthermore, the effect observed in the tail thrombosis model might be explained completely by increased vasodilation. These results indicate that curdione may be one of the main bioactive constituents in Rhizoma Curcumae that removes blood stasis.     

CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the protein Huntingtin (Htt). We previously reported that mutant Htt expression activates the ERK1/2 and JNK pathways [Apostol, B.L., Illes, K., Pallos, J., Bodai, L., Wu, J., Strand, A., Schweitzer, E.S., Olson, J.M., Kazantsev, A., Marsh, J.L., Thompson, L.M., 2006. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. 15, 273-285]. Chemical and genetic modulation of these pathways promotes cell survival and death, respectively. Here we test the ability of two closely related compounds, CEP-11004 and CEP-1347, which inhibit Mixed Lineage Kinases (MLKs) and are neuroprotective, to suppress mutant Htt-mediated pathogenesis in multiple model systems. CEP-11004/CEP-1347 treatment significantly decreased toxicity in mutant Htt-expressing cells that evoke a strong JNK response. However, suppression of cellular dysfunction in cell lines that exhibit only mild Htt-associated toxicity and little JNK activation was associated with activation of ERK1/2. These compounds also reduced neurotoxicity in immortalized striatal neurons from mutant knock-in mice and Drosophila expressing a mutant Htt fragment. Finally, CEP-1347 improved motor performance in R6/2 mice and restored expression of BDNF, a critical neurotrophic factor that is reduced in HD. These studies suggest a novel therapeutic approach for a currently untreatable neurodegenerative disease, HD, via CEP-1347 up-regulation of BDNF.