Design and Biological Evaluation of the Long-Acting C5-Inhibited Ornithodoros moubata Complement Inhibitor (OmCI) Modified with Fatty Acid.

Disorder of complement response is a significant pathogenic factor causing some autoimmune and inflammation diseases. The Ornithodoros moubata Complement Inhibitor (OmCI), a small 17 kDa natural protein, was initially extracted from soft tick salivary glands. The protein was found binding to complement C5 specifically, inhibiting the activation of the complement pathway, which is a successful therapeutic basis of complement-mediated diseases. However, a short half-life due to rapid renal clearance is a common limitation of small proteins for clinical application. In this study, we extended the half-life of OmCI by modifying it with fatty acid, which was a method used to improve the pharmacokinetics of native peptides and proteins. Five OmCI mutants were initially designed, and single-site cysteine mutation was introduced to each of them. After purification, four OmCI mutants were obtained that showed similar in vitro biological activities. Three mutants of them were subsequently coupled with different fatty acids by nucleophilic substitution. In total, 15 modified derivatives were screened and tested for anticomplement activity in vitro. The results showed that coupling with fatty acid would not significantly affect their complement-inhibitory activity (CH50 and AH50). OmCIT90C-CM02 and OmCIT90C-CM05 were validated as the applicable OmCI bioconjugates for further pharmacokinetic assessments, and both showed improved plasma half-life in mice compared with unmodified OmCI (15.86, 17.96 vs 2.57 h). In summary, our data demonstrated that OmCI conjugated with fatty acid could be developed as the potential long-acting C5 complement inhibitor in the clinic.

A new monoterpene glycoside from Pedicularis verticillata and anticomplementary activity of its compounds.

A new monoterpene glycoside named as pedivertoside D (1), together with 13 known compounds (2-14, resp.) were isolated from the whole plant of Pedicularis verticillata L. The new compound was identified as (2E,6E,5R)-5,8-dihydrooxy-2,6-dimethyl-3,7-octadienyl-ß-D-glucopyranoside by spectroscopic methods including 2 D-NMR techniques. The known compounds were determined spectroscopically and compared with previously reported spectral data. Compounds 6 and 9 exhibited anticomplementary effects against the classical pathway (CP) with CH50 values of 0.07 mM and 0.23 mM, respectively, which are plausible candidates for developing potent anti-complementary agents from this plant.

Juniperus pingii var. wilsonii acidic polysaccharide: Extraction, characterization and anticomplement activity.

A water-soluble acidic polysaccharide, XB-PS3, was isolated from the twigs of Juniperus pingii var. Wilsonii with a molecular weight of 86.04 kDa. By means of monosaccharide composition analysis, methylation, 2D NMR spectroscopy and UPLC-MS analysis, we concluded that XB-PS3 had a backbone composed of →2,4)-α-Manp-(1→ and →4)-α-GalpA-(1→ (60 % esterified), with an araban branch attached to O-2 of →2,4)-α-Manp-(1→. The possible repeating units were further validated by oligosaccharide analysis and partial acid hydrolysis. XB-PS3 exhibited potent anticomplement activity with CH50 value of 117.23 ± 18.74 µg/mL and interacted with C3, C4, C5 and C9 in the complement activation cascade. However, the anticomplement activity was significantly weakened when the galacturonic acids were reduced (CH50: 268.55 ± 16.82 µg/mL) or the branches were removed by partial hydrolysis (CH50: 197.76 ± 21.81 µg/mL), indicating the important role of uronic acids and branch structure in the polysaccharide's anticomplement activity.

Quassinoids from seeds of Brucea Javanica and their anticomplement activities.

An investigation on seeds of Brucea javanica led to the acquisition of a new quassinoid, 20-hydroxyyadanzigan (1), along with five known quassinoids (2-6). The structure of the new compound was elucidated on the basis of extensive spectral analysis. All of the compounds were assayed for their anticomplement activities through classical and alternative pathways. Compounds 1-6 exhibited potent anticomplement activity with CH50 and AP50 values of 0.032-0.075 mg/mL and 0.061-0.118 mg/mL, respectively. Moreover, the structure-activity relationships of these compounds are discussed.[Formula: see text].

Anticomplement and antitussive activities of major compound extracted from Chimonanthus nitens Oliv. leaf.

Chimonanthus nitens Oliv. leaf (CNOL), as a traditional Chinese medicine, has been widely used for the treatment of influenza and colds over a long history. However, the mechanism of colds related to the effects of CNOL have been little studied. In this study, the anticomplement and antitussive activities of different polarity extracts of CNOL were evaluated. Ethyl acetate extract (EAE) among different extracts not only significantly decreased cough times by 21-58% (P < 0.01), but also had anticomplement effects demonstrated by the CH50 values of 0.100 mg/ml. A total of 28 constituents (10 coumarins, 13 flavonoids and five phenolics) were identified in EAE based on the ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry technique. Eight compounds in EAE were evaluated by an ammonia-induced cough model to reveal the antitussive mechanisms and classical anticomplement pathway. The results indicated that the antitussive effects of scopoletin, kaempferol-3-O-rutinoside and kaempferol may depend on central mechanisms and that flavonoids such as compounds of kaempferol-3-O-rutinoside and kaempferol have better anticomplementary activity than coumarins like compounds of scopolin, scopoletin and isofraxidin. Taken together, kaempferol-3-O-rutinoside and kaempferol could be important chemical markers in the present study that might be used to evaluate the quality and biological activity of CNOL.

Screening of anti-complement active ingredients from Eucommia ulmoides Oliv. branches and their metabolism in vivo based on UHPLC-Q-TOF/MS/MS.

Eucommia ulmoides Oliv. (E. ulmoides) is a kind of plant with high medicinal value, there are known as the "gold plants". Some components and contents of barks and branches from E. ulmoides are similar, the barks are mainly used as medicine, but the branches have not been systematically studied and were discarded. In this paper, five fractions extracted from E. ulmoides branches were detected by the classical anti-complement activity assay in vitro. The n-butanol fraction of E. ulmoides branches showed excellent anti-complement activities with a CH50 value of 0.016 ±â€¯0.0014 mg·mL-1. A total of 76 compounds were identified from the n-butanol fraction, including 9 alkaloids, 18 organic acids, 22 lignans, 15 phenylethanoid glycosides and 12 other compounds. To further prove the anti-complement activity of potential active compounds, those compounds detectable in rat plasma after oral administration were tested by classical anti-complement activity assays. Genipin and pinoresinol 4-O-glucopyranoside had a certain complement inhibitory activity in the 17 potential anti-complements, their CH50 values were 0.050 ±â€¯0.0038 and 0.022 ±â€¯0.0018 mg·mL-1. UHPLC-Q-TOF/MS/MS was developed to profile and characterize the metabolites of genipin and pinoresinol 4-O-glucopyranoside in rat plasma. Twenty-one and seventeen metabolites were found, respectively. In summary, this study reported important clues for the further pharmacological and clinical studies of E. ulmoides branches. Meanwhile, it provided a practical strategy for rapid screening and identifying of in vivo anti-complement in traditional Chinese medicine.

A novel dimeric flavonol glycoside from Cynanchum acutum subsp. sibiricum.

A novel dimeric flavonol glycoside, Cynanflavoside A (1), together with six analogues, kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranoside (2), quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranoside (3), kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-ß-D-xylopyranoside (4), quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-ß-D-xylopyranoside (5), kaempferol-3-O-ß-D-glucopyranosyl-7-O-α-L-rhamnopyranoside (6), and quercetin-3-O-galactoside (7) were isolated from the n-butyl alcohol extract of Cynanchum acutum subsp. sibiricum. Their structures were determined spectroscopically and compared with previously reported spectral data. All compounds were evaluated for their anti-complementary activity in vitro, and only compound 5 exhibited anti-complement effects with CH50 value of 0.33 mM.

Developments in anti-complement therapy; from disease to clinical trial.

The complement system is well known for its role in innate immunity and in maintenance of tissue homeostasis, providing a first line of defence against infection and playing a key role in flagging apoptotic cells and debris for disposal. Unfortunately complement also contributes to pathogenesis of a number of diseases; in some cases driving pathology, and in others amplifying or exacerbating the inflammatory and damaging impact of non-complement disease triggers. The role of complement in pathogenesis of an expanding number of diseases has driven industry and academia alike to develop an impressive arsenal of anti-complement drugs which target different proteins and functions of the complement cascade. Evidence from genetic and biochemical analyses, combined with improved identification of complement biomarkers and supportive data from sophisticated animal models of disease, has driven a drug development landscape in which the indications selected for clinical trial cluster in three 'target' tissues: the kidney, eye and vasculature. While the disease triggers may differ, complement activation and amplification is a common feature in many diseases which affect these three tissues. An abundance of drugs are in clinical development, some show favourable progression whereas others experience significant challenges. However, these hurdles in themselves drive an ever-evolving portfolio of 'next-generation' drugs with improved pharmacokinetic and pharmacodynamics properties. In this review we discuss the indications which are in the drug development 'spotlight' and review the relevant indication validation criteria. We present current progress in clinical trials, highlighting successes and difficulties, and look forward to approval of a wide selection of drugs for use in man which give clinicians choice in mechanistic target, modality and route of delivery.

Chemical characterization and complement modulating activities of an arabinogalactan-protein-rich fraction from an aqueous extract of avocado leaves.

The aim of this study was to chemically characterize an arabinogalactan-protein-rich fraction (FRAGP) obtained from an aqueous extract of avocado leaves and investigate its effects on the classical pathway of the complement system. The FRAGP contained 4.6% ±â€¯1.8%, 22.5% ±â€¯4.9%, and 76.7% ±â€¯8.8% of total protein, arabinogalactan-protein, and carbohydrates, respectively. Arabinose and galactose were the main monosaccharide constituents. FT-IR and NMR data, together with linkage analyses, indicated the presence of a structure that included a (1 → 3)-linked ß-D-Galp main chain, mainly substituted at O-6 by Gal and Ara residues, which was characteristic of a type II arabinogalactan. The effect of FRAGP on the classical pathway of complement system was examined by a hemolytic fixation test and comparing with heparin, which was used as a control for inhibition. With pre-incubation, the IC50 of FRAGP was 1.90 ±â€¯1.1 µg/mL, which was similar to that of heparin (IC50 = 2.90 ±â€¯0.3 µg/mL). Without pre-incubation, the IC50 values were 18.6 ±â€¯3.7 and 8.0 ±â€¯4.1 µg/mL for FRAGP and heparin, respectively. Collectively, these results suggested that FRAGP has an inhibitory effect on the classical pathway of the complement system.

Anticomplement Treatment in Atypical and Typical Hemolytic Uremic Syndrome.

The dissection of the pathogenic mechanisms of the various forms of the hemolytic uremic syndrome (HUS) has paved the way for the design of specific efficacious treatments. Such mechanistic approach led to a revolution in the management of atypical HUS with the use of the first-in class C5 blocker, eculizumab. The availability of this anticomplement drug has also raised unsettled questions regarding the cost or burden and optimal duration of therapy and its use in secondary HUS. The efficacy of eculizumab in Shiga toxin producing Escherichia coli-associated HUS is not to date established and the results of ongoing prospective studies are eagerly awaited. Nevertheless, the emergence of anticomplement therapies (eculizumab and other drugs in development) has transformed our approach of HUS.

Anticomplement compounds from Polygonum chinense.

Five new compounds including two phenyldilactones (1, 2), two coumarins (3, 4) and a dimer of N-E-feruloyl tyramine (5) together with twenty-three known compounds (6-28) were isolated from a medicinal plant Polygonum chinense. The structures of the new compounds were established by detailed spectral analysis. The absolute configurations of 1 and 5 were elucidated by Mosher's method, Mo2(OAc)4-induced electronic circular dichroism (ECD) data, and ECD calculation. All the compounds were found to show potent anticomplement activity with CH50 and AP50 values ranging from 0.18 to 1.45 mM, and 0.26 to 2.80 mM, respectively. Phenyldilactones and phenylpropionic tyramines were firstly reported as anticomplement agents. The targets of compounds 1, 3, 5 and 10 in complement activation cascade were identified as well.

Acetylcholine receptor antibody-mediated animal models of myasthenia gravis and the role of complement.

Because of the failure of many promising therapeutics identified in preclinical evaluation, funding sources have established guidelines for increased rigor in animal evaluations. The myasthenia gravis (MG) community of scientists has developed guidelines for preclinical assessment for potential MG treatments. Here, we provide a focused summary of these recommendations and the role of complement in disease development in experimental models of MG.

Inhibition of Complement Drives Increase in Early Growth Response Proteins and Neuroprotection Mediated by Salidroside After Cerebral Ischemia.

Salidroside is neuroprotective across a wide therapeutic time-window after cerebral ischemia-reperfusion injury (IRI). Here, we investigated the role of complement in mediating effects of salidroside after cerebral IRI in rats. Rats were administrated with vehicle or salidroside 50 mg/kg, given daily for either 24 or 48 h, after middle cerebral artery occlusion (MCAO) for 2 h and reperfusion for 1 h. Levels of proteins in ischemic brain were measured by immunofluorescence and western blotting. We observed early increases in the deposition of immunoglobulin M, mannose-binding lectin 2, and annexin IV on cerebral endothelial cells, induction of the complement components C3 and C3a, by 24 h after IRI, and a later significant increase in the complement component C1q by 48 h. Salidroside prevented these changes. The neuroplasticity-related early growth response proteins Egr1, Egr2, and Egr4 and activity-regulated cytoskeleton-associated protein increased transiently in the first 6 h after IRI but then decreased below baseline by 48 h after IRI. Neither salidroside nor a C3a receptor antagonist (C3aRA) affected these proteins 24 h after IRI, but both reversed their later decreases to similar and non-additive extents. Salidroside and C3aRA increased NeuN in a non-additive manner after IRI. Our results suggest that salidroside exerts neuroprotection by reducing early activation of the lectin pathway on the cerebral endothelium and inhibiting the gradual activation of the classical pathway after cerebral IRI. This prolonged neuroprotection may depend, at least in part, on increased expression of neuroplasticity-related genes driven by reduced complement activation.

Anti-complementary constituents of Anchusa italica.

Activity-guided fractionation for complement inhibitors led to the isolation of 24 known compounds from Anchusa italica. Chemical types include eight megastigmane compounds, five triterpenoid compounds, five lignan compounds, three flavonoid compounds, two alkaloid compounds and one phenthyl alcohol compound. Among which, a lignan (medioresinol), an alkaloid (5-hydroxypyrrolidin-2-one) and a flavonoid (5-hydroxyl-3', 4', 6, 7-tetramethoxy flavone) exhibited better anticomplementary effects with CH50 values ranging from 0.07 to 0.82 mM, which are plausible candidates for developing potent anticomplementary agents.

Anticomplement triterpenoids from the roots of Ilex asprella.

Five new (1-5) and twenty-eight known (6-33) triterpenoids were isolated from the roots of Ilex asprella. The structures of the new compounds were elucidated by the detailed spectral analysis. The ursane and oleanane triterpenoids were found to show anticomplement activity with some structure-activity relationships. Several triterpenoids (1-3, 6-7) exhibited potent anticomplement activity with the CH50 and AP50 values of 0.058-0.131mg/mL and 0.080-0.444mg/mL, respectively. It was found that caffeoyl group could enhance activity remarkably, followed by coumaroyl and feruloyl group. The 28-carboxyl group was also important to anticomplement activity for the triterpenoids. However, the triterpenoids with lactone ring (4, 9-14) exhibited weak activity and triterpenoid glycosides (5, 23-33) showed no inhibition. The targets of several bioactive triterpenoids in complement activation cascade were identified as well.

Structure analysis of a heteropolysaccharide from Taraxacum mongolicum Hand.-Mazz. and anticomplementary activity of its sulfated derivatives.

A homogenous water-soluble polysaccharide, DPSW-A, with a deduced chemical structure was extracted from the herb Taraxacum mongolicum Hand.-Mazz. Moreover, 80.813-kDa DPSW-A is composed of three types of monosaccharide, namely rhamnose, arabinose, and galactose, at a molar ratio of 1.0:10.7:11.9. The main chain of DPSW-A contains Terminal-Galp, 1,3-Galp, 1,6-Galp, 1,3,6-Galp, and 1,2,4-Rhap; the branched chain contains Terminal-Araf, 1,5-Araf, and 1,3,5-Araf. The sulfated derivatives prepared from DPSW-A showed inhibitory effects on complement activation through the classical pathway (CH50: Sul-DPSW-A, 3.94±0.43µg/mL; heparin, 104.40±3.82µg/mL) and alternative pathway (AP50: Sul-DPSW-A, 42.76±0.46µg/mL; heparin, 43.42±0.22µg/mL). Mechanism studies indicated that Sul-DPSW-A inhibited complement activation by blocking C1q, C1r, C1s, and C9, but not C2, C3, C4, and C5. In addition, Sul-DPSW-A displayed limited anticoagulant effects. These results suggest that Sul-DPSW-A prepared from DPSW-A is valuable for treating diseases caused by excessive complement system activation.

Cycloartane triterpenes from Beesia calthaefolia and their anticomplement structure-activity relationship study.

Fifteen cycloartane triterpenes were isolated from Beesia calthaefolia and among them one was new cycloartane triterpenoid. The structure of new compound was determined by the application of spectroscopic analyses and chemical methods. The fifteen compounds were evaluated for their anticomplement activity by classic pathway. The structure-activity relationship analysis indicated that the configurations of 12-OH is preferable to be α than ß, and 18-OH can decrease while 15-OH can increase the anticomplement activity, but saponin with both 15-OH and 18-OH lost most of its activity. The glycosyl moiety of most isolated cycloartane triterpenes is xylosyl. When xylosyl was substituted by glucosyl or galactosyl, their anticomplement activities were decreased or increased, respectively. Further structure-activity relationship (SAR) studies must be carried out to achieve general conclusions regarding the effect of further functionalizations on the anticomplement saponins.

One new cycloartane triterpene glycoside from Beesia calthaefolia.

One new cycloartane triterpene glycoside (1) was isolated from the whole plant of Beesia calthaefolia. Its structure was elucidated on the basis of extensive spectroscopic data analysis. Its inhibitory effect was measured by the classical pathway of the complement system, and compared with those of known related cycloartane glycosides 2 and 3, previously isolated by us from the same plant. Compounds 1 and 2 exhibited inhibitory activity of complement system with IC50 of 395.3 and 214 µM, respectively. The results suggested that OH at C-12, C-18 and C-15 along with the polarity could affect the inhibitory activity.

Prevention of Defective Placentation and Pregnancy Loss by Blocking Innate Immune Pathways in a Syngeneic Model of Placental Insufficiency.

Defective placentation and subsequent placental insufficiency lead to maternal and fetal adverse pregnancy outcome, but their pathologic mechanisms are unclear, and treatment remains elusive. The mildly hypertensive BPH/5 mouse recapitulates many features of human adverse pregnancy outcome, with pregnancies characterized by fetal loss, growth restriction, abnormal placental development, and defects in maternal decidual arteries. Using this model, we show that recruitment of neutrophils triggered by complement activation at the maternal/fetal interface leads to elevation in local TNF-α levels, reduction of the essential angiogenic factor vascular endothelial growth factor, and, ultimately, abnormal placentation and fetal death. Blockade of complement with inhibitors specifically targeted to sites of complement activation, depletion of neutrophils, or blockade of TNF-α improves spiral artery remodeling and rescues pregnancies. These data underscore the importance of innate immune system activation in the pathogenesis of placental insufficiency and identify novel methods for treatment of pregnancy loss mediated by abnormal placentation.

Ischemia/reperfusion injury: effect of simultaneous inhibition of plasma cascade systems versus specific complement inhibition.

Ischemia/reperfusion injury (IRI) may occur from ischemia due to thrombotic occlusion, trauma or surgical interventions, including transplantation, with subsequent reestablishment of circulation. Time-dependent molecular and structural changes result from the deprivation of blood and oxygen in the affected tissue during ischemia. Upon restoration of blood flow a multifaceted network of plasma cascades is activated, including the complement-, coagulation-, kinin-, and fibrinolytic system, which plays a major role in the reperfusion-triggered inflammatory process. The plasma cascade systems are therefore promising therapeutic targets for attenuation of IRI. Earlier studies showed beneficial effects through inhibition of the complement system using specific complement inhibitors. However, pivotal roles in IRI are also attributed to other cascades. This raises the question, whether drugs, such as C1 esterase inhibitor, which regulate more than one cascade at a time, have a higher therapeutic potential. The present review discusses different therapeutic approaches ranging from specific complement inhibition to simultaneous inhibition of plasma cascade systems for reduction of IRI, gives an overview of the plasma cascade systems in IRI as well as highlights recent findings in this field.