Recombinant Inhibitor Research Articles

Recombinant Brugia malayi pepsin inhibitor (rBm33) exploits host signaling events to regulate inflammatory responses associated with lymphatic filarial infections

Prolonged existence of filarial parasites and their molecules within the host modulate the host immune system to instigate their survival and induce inflammatory responses that contribute to disease progression. Recombinant Brugia malayi pepsin inhibitor (rBm33) modulates the host immune responses by skewing towards Th1 responses characterized by secretion of inflammatory molecules such as TNF-α, IL-6, nitric oxide (NO). Here we also specified the molecular signaling events triggered by rBm33 in peripheral blood mononuclear cells (PBMCs) of filarial endemic normals (EN). rBm33 predominantly enhanced the levels of nitric oxide in cultured PBMCs but did not result in oxidative stress to the host cells. Further, rBm33 treatment of human PBMCs resulted in higher GSH/GSSG levels. MYD88 dependent activation was found to be associated with rBm33 specific inflammatory cytokine production. rBm33 triggered intracellular signaling events also involved JNK activation in host PBMCs. In addition, c-Fos and not NF-κB was identified as the transcription factor regulating the expression of inflammatory cytokines in rBm33 stimulated PBMCs. rBm33 marked its role in filarial pathology by altered levels of growth factors but did not have a significant impact on matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) activity of host PBMCs. Thus, the study outlines the signaling network of rBm33 induced inflammatory responses within the host immune cells.

Efficacy of recombinant human C1 esterase inhibitor for the treatment of severe hereditary angioedema attacks.

Severe attacks of hereditary angioedema (HAE) are debilitating and potentially life threatening, and can increase anxiety and the use of medical resources. This post hoc assessment evaluated recombinant human C1 esterase inhibitor (rhC1-INH) used to treat acute severe HAE attacks. In a double-blind, randomized-controlled trial (RCT), patients with an HAE attack (baseline visual analog scale score of ≥50 mm, with severe attacks defined as ≥75 mm) were randomly assigned to receive rhC1-INH (50 IU/kg for patients who weighed <84 kg; 4200 IU for patients who weighed ≥84 kg) or placebo. Also, in an open-label extension (OLE) study of rhC1-INH, oropharyngeal-laryngeal attacks were analyzed. Rescue therapy with rhC1-INH 50 IU/kg (≤4200 IU) was permitted after 4 hours or for life-threatening symptoms (in the RCT) or after 1 hour (in the OLE study). The primary end point measured the time to the beginning of symptom relief by using the Treatment Effects Questionnaire. Of 75 adults in the RCT, 43 had severe attacks and received either rhC1-INH (n = 24) or placebo (n = 19). The median (95% confidence interval) time to the onset of symptom relief totaled 90.0 minutes (95% confidence interval, 47.0-120.0 minutes) versus 334.0 minutes (95% confidence interval, 105.0 to not calculable minutes; hazard ratio, 2.5; p = 0.02), for rhC1-INH and placebo, respectively. Open-label rhC1-INH rescue therapy was administered to 1 of 24 in the rhC1-INH group (4.2%) and 10 of 19 in the placebo group (52.6%). During the OLE study, the median onset of symptom relief with rhC1-INH for eight oropharyngeal-laryngeal HAE attacks was 69.0 minutes (95% confidence interval, 59.0-91.0 minutes). In the current study, rhC1-INH was efficacious in resolving severe HAE attacks, including oropharyngeal-laryngeal attacks. The rhC1-INH rescue treatment rapidly improved symptoms for patients who received placebo and who experienced worsening or sustained symptoms.

Experimental investigation of photocatalytic and photovoltaic activity of titania/rice husk crystalline nano-silica hybrid composite

As a kind of highly operative, stable photocatalysts and low‐cost, TiO2 has received significant scientific attention. However, it can only be initiated under ultraviolet light irradiation owing to its wide bandgap, weak separation efficiency of carriers and high recombination. Doping or making composites is an active method to outspread the light absorption to the visible light region. This article describes the extraction procedure of nano-silica from rice husk through a dissolution-precipitation process and producing titania-nanosilica composites by sol-gel method for photocatalytic and photovoltaic application where extracted nanosilica act as green filler to enhance the surface area of titania photocatalyst and an inhibitor of recombination. Our general plans are anticipated by passivated composite to enhance the activity of TiO2 semiconductors. This results show that the TiO2 /rice husk extracted crystalline nanosilica (RHCNS) has formed an active hybrid photocatalyst which is significant for photovoltaics application also. Large enhancement of photovoltaic parameters and rate of dye degradation of the TiO2/RHCNS composite observed which is stepping path to further development in adsorption-assisted photocatalysts under both UV and visible lights.

Aviscumine, a recombinant ribosomal inhibitor, increases the antitumor activity of natural killer cells.

Aviscumine, a recombinant lectin I, has been identified as an immunomodulatory agent within a new class of ribotoxic stress-inducing anticancer substances that have demonstrated efficacy in phase I/II trials. The aim of the present study was to elucidate the presumed effect of aviscumine on enhancing human natural killer (NK) cell antitumor cytotoxicity. To measure the effect of aviscumine on human NK cell cytotoxicity, chromium-51-release assays against K-562 cells were performed with isolated NK cells from the whole blood of 34 healthy volunteers. Two effector-to-target cell ratios (12.5:1 and 25:1) were used by two independent investigators with a focus on the concentration-dependent effect (0.5 vs. 1 ng/ml aviscumine), reproducibility (first vs. second investigator) and the specificity of the effect by comparison to a heat-inactivated aliquot and interleukin 2 (IL-2) stimulation (10 ng/ml). The mediation of the effect via degranulation was demonstrated by flow cytometric analyses of CD107α expression. Statistics were performed with SPSS using Student's t-tests for normally distributed data. Aviscumine induced a significant and reproducible, concentration-dependent increase in NK cell cytotoxicity (n=22; P<0.01 for both concentrations and ratios), which was also demonstrated when administered in combination with IL-2 (n=12; 12.5:1 ratio, P<0.001; 25:1 ratio, P=0.025) and when compared with the heat-inactivated aliquots (n=12; 12.5:1, P=0.004; 25:1 ratio, P=0.007). The mediation of its effect via interferon γ degranulation was demonstrated by significantly enhanced CD107α expression (n=7; P=0.005). Taken together, the results indicate that aviscumine induced an increase in NK cell anticancer cytotoxicity. These results highlight its clinical potential as an immunostimulatory agent, particularly with regard to combined use with chemotherapeutics or immune checkpoint inhibitors. However, further studies are required.

Recombinant human C1 esterase inhibitor for prophylaxis of hereditary angio-oedema: a phase 2, multicentre, randomised, double-blind, placebo-controlled crossover trial

Hereditary angio-oedema is a recurrent, oedematous disorder caused by deficiency of functional C1 inhibitor. Infusions of plasma-derived C1 esterase inhibitor deter attacks of hereditary angio-oedema, but the prophylactic effect of recombinant human C1 esterase inhibitor has not been rigorously studied. We aimed to assess the efficacy of recombinant human C1 esterase inhibitor for prophylaxis of hereditary angio-oedema. We conducted this phase 2, multicentre, randomised, double-blind, placebo-controlled crossover trial at ten centres in Canada, the Czech Republic, Israel, Italy, Macedonia, Romania, Serbia, and the USA. We enrolled patients aged 13 years or older with functional C1-inhibitor concentrations of less than 50% of normal and a history of four or more attacks of hereditary angio-oedema per month for at least 3 months before study initiation. Patients were randomly assigned centrally (1:1:1:1:1:1), via an interactive response technology system with fixed allocation, to receive one of six treatment sequences. During each sequence, patients received intravenous recombinant human C1 esterase inhibitor (50 IU/kg; maximum 4200 IU) twice weekly, recombinant human C1 esterase inhibitor once weekly and placebo once weekly, and placebo twice weekly, each for 4 weeks with a 1 week washout period between crossover. All patients, investigators, and study personnel who participated in patient care were masked to group allocation during the study. The primary efficacy endpoint was the number of attacks of hereditary angio-oedema observed in each 4 week treatment period. Attack symptoms were recorded daily. The primary efficacy analysis was done in the intention-to-treat population. Safety was assessed in all patients who received at least one injection of study medication. This study is registered with ClinicalTrials.gov, number NCT02247739. Between Dec 29, 2014, and May 3, 2016, we enrolled 35 patients, of whom 32 (91%) underwent randomisation (intention-to-treat population) and 26 (81%) completed the study. The mean number of attacks of hereditary angio-oedema over 4 weeks was significantly reduced with recombinant human C1 esterase inhibitor twice weekly (2·7 attacks [SD 2·4]) and once weekly (4·4 attacks [3·2]) versus placebo (7·2 attacks [3·6]), with mean differences of -4·4 attacks (p<0·0001) and -2·8 attacks (p=0·0004), respectively. We recorded adverse events in ten (34%) of 29 patients given twice-weekly recombinant human C1 esterase inhibitor, 13 (45%) of 29 patients given the once-weekly regimen, and eight (29%) of 28 patients given placebo. Headache (twice-weekly treatment) and nasopharyngitis (once-weekly treatment) were the most common adverse events. Two (7%) adverse events (fatigue and headache) were deemed possibly related to treatment with recombinant human C1 esterase inhibitor, but both resolved without additional treatment. No thrombotic or thromboembolic events, systemic allergic reactions (including anaphylaxis), or neutralising antibodies were reported. Prophylaxis with recombinant human C1 esterase inhibitor provided clinically relevant reductions in frequency of hereditary angio-oedema attacks and was well tolerated. In view of the pharmacokinetic profile of recombinant human C1 esterase inhibitor, our results suggest that efficacy of C1-inhibitor replacement therapy might not be a direct function of plasma trough concentrations of C1 inhibitor. Pharming Technologies.

Rapid, Automated, and Specific Immunoassay to Directly Measure Matrix Metalloproteinase-9-Tissue Inhibitor of Metalloproteinase-1 Interactions in Human Plasma Using AlphaLISA Technology: A New Alternative to Classical ELISA.

The protocol describes a novel, rapid, and no-wash one-step immunoassay for highly sensitive and direct detection of the complexes between matrix metalloproteinases (MMPs) and their tissue inhibitor of metalloproteinases (TIMPs) based on AlphaLISA® technology. We describe two procedures: (i) one approach is used to analyze MMP-9–TIMP-1 interactions using recombinant human MMP-9 with its corresponding recombinant human TIMP-1 inhibitor and (ii) the second approach is used to analyze native or endogenous MMP-9–TIMP-1 protein interactions in samples of human plasma. Evaluating native MMP-9–TIMP-1 complexes using this approach avoids the use of indirect calculations of the MMP-9/TIMP-1 ratio for which independent MMP-9 and TIMP-1 quantifications by two conventional ELISAs are needed. The MMP-9–TIMP-1 AlphaLISA® assay is quick, highly simplified, and cost-effective and can be completed in less than 3 h. Moreover, the assay has great potential for use in basic and preclinical research as it allows direct determination of native MMP-9–TIMP-1 complexes in circulating blood as biofluid.

Sensitivity enhancement of chemically amplified EUV resists by adding acid-generating promoters

The trade-off relationship between sensitivity, resolution, and roughness is a serious problem in the development of extreme ultraviolet (EUV) resist materials. Increasing the acid-generation efficiency is an effective solution to this trade-off problem. Chemically amplified resists (CARs) remain important for EUV lithography because of their longstanding use in photolithography. In this study, the feasibility and mechanism of improving CAR performance by adding acid-generating promoters were investigated. It was confirmed that di-p-tolyl sulfone acts as both a deprotonation promoter and a recombination inhibitor, which enhances the acid-generation efficiency and improves the performance of CARs in lithographies using ionizing radiation such as EUV and electron beams.

Targeting the Bcl-2 family and P-glycoprotein reverses paclitaxel resistance in human esophageal carcinoma cell line

Paclitaxel (PTX) is one of the most effective drugs used in the treatment of esophageal cancer, however, paclitaxel resistance represents a key limitation during the treatment process. In this study, we investigated the changes of Bcl-2 family members in the moderate paclitaxel-resistance of esophageal carcinoma EC109/PTX cells both in vitro and in vivo. Moreover, we evaluated the reversal effect using siRNAs and the recombinant inhibitor TW37 targeting Bcl-2, Bcl-XL and Mcl-1. Our findings show that downregulation of Bcl-2, Bcl-XL and Mcl-1 can significantly promote EC109/PTX cell apoptosis and reduce the EC109/PTX cell resistance index (RI). Furthermore, TW37 in combination with a P-gp inhibitor can synergistically reverse the paclitaxel resistance in EC109/PTX cells. These results suggest that targeting of the Bcl-2 family and P-gp is capable of reversing the resistance in EC109/PTX cells and the two-inhibitor combination may be a novel treatment strategy for resistant esophageal cancer.

In Vitro Metabolism of Oprozomib, an Oral Proteasome Inhibitor: Role of Epoxide Hydrolases and Cytochrome P450s.

Oprozomib is an oral proteasome inhibitor currently under investigation in patients with hematologic malignancies or solid tumors. Oprozomib elicits potent pharmacological actions by forming a covalent bond with the active site N-terminal threonine of the 20S proteasome. Oprozomib has a short half-life across preclinical species and in patients due to systemic clearance via metabolism. Potential for drug-drug interactions (DDIs) could alter the exposure of this potent therapeutic; therefore, a thorough investigation of pathways responsible for metabolism is required. In the present study, the major drug-metabolizing enzyme responsible for oprozomib metabolism was identified in vitro. A diol of oprozomib was found to be the predominant metabolite in human hepatocytes, which formed via direct epoxide hydrolysis. Using recombinant epoxide hydrolases (EHs) and selective EH inhibitors in liver microsomes, microsomal EH (mEH) but not soluble EH (sEH) was found to be responsible for oprozomib diol formation. Coincubation with 2-nonylsulfanyl-propionamide, a selective mEH inhibitor, resulted in a significant decrease in oprozomib disappearance (>80%) with concurrent complete blockage of diol formation in human hepatocytes. On the contrary, a selective sEH inhibitor did not affect oprozomib metabolism. Pretreatment of hepatocytes with the pan-cytochrome P450 (P450) inhibitor 1-aminobenzotriazole resulted in a modest reduction (∼20%) of oprozomib metabolism. These findings indicated that mEH plays a predominant role in oprozomib metabolism. Further studies may be warranted to determine whether drugs that are mEH inhibitors cause clinically significant DDIs with oprozomib. On the other hand, pharmacokinetics of oprozomib is unlikely to be affected by coadministered P450 and sEH inhibitors and/or inducers.

Recombinant human Tissue Inhibitor of Metalloprotease −2; a novel anti‐cancer therapeutic

INTRODUCTIONTissue inhibitor of Metalloprotease‐2 (TIMP‐2), a soluble endogenous ~22 kDa metalloprotease inhibitor, exhibits anti‐cancer properties by metalloprotease inhibition in tumor microenvironment, direct receptor‐mediated growth inhibition, increased chemo –sensitivity and decreased cell motility. Herein we report a complete production‐characterization process, biophysical analysis, anti‐MMP activity and anti‐proliferative activity of rhTIMP‐2 in cell lines and mouse models of breast and lung cancer. Further we report the development and characterization of rhTIMP‐2 extended release peptide hydrogels and desferoxamine(des) labelled rhTIMP‐2 for dosage form and PET scan probe development respectively.METHODSHuman TIMP‐2 cDNA with 6xHistidine epitope tag was codon optimized, transfected to HEK 293F cells and grown in optimized conditions up to 5L scale in wave bags. rhTIMP‐2 was captured by Ni2+ IMAC, polished by preparative RP‐HPLC and formulated in pure MQ water. rhTIMP‐2 was analyzed by SDS‐PAGE, Western Blotting, ESI‐TOF mass spec and CD spectroscopy. rhTIMP‐2 was also studied by 2D HSQC NMR and by Isothermal titration calorimetry for binding to integrin alpha‐3: beta‐1 receptor ectodomain both at 37°C. All production batches were Endotoxin and MAP tested. In‐vitro MMP‐2 inhibitory and A549 cancer cell growth inhibitory assay was done by colorimetric plate‐based methods. In‐vivo rhTIMP‐2 was administered i.p. to athymic nude mice bearing breast cancer tumors against HBSS controls.RESULTSCodon optimization and stable clonal selection resulted in about 7‐fold increase in rhTIMP‐2 His6 expression compared to identical culture with WT rTIMP‐2 cDNA transfected HEK‐293F cells. Purification by IMAC Ni2+ chromatography and RP‐HPLC yielded TIMP‐2 His6 with greater than 90% (96.21% recovery) and 99% (81.75% recovery) percentage purities respectively. Purified rTIMP‐2 exhibited single band and peak in SDS‐PAGE gel and by ESI‐TOF mass spec.; and was immuno‐reactive against anti‐TIMP‐2 and poly‐histidine antibodies. Circular Dichorism spectra indicated high degree of β‐pleated sheet in rhTIMP‐2 2° structure. 2D NMR of rhTIMP‐2 His6 in natural abundance indicated a well folded monodisperse rhTIMP‐2 with ~10–15% population of alternate rhTIMP‐2 species. All production batches of the rTIMP‐2 were negative in MAP testing with Endotoxin levels at 3.702 EU/ml (&lt;10 EU/ml acceptable in cGMP). TIMP‐2 effectively displayed dose dependent inhibition of MMP‐2 (type IV collagenase) activity with an IC50 of &lt; 50nM; with no MMP‐2 inhibitory observed in the Ala+TIMP‐2 (N′ terminal Ala appended TIMP‐2 with no MMP inhibitory activity). In‐vitro pre‐treatment with 50 nM rTIMP‐2 in gelatin coated plates suppressed rhEGF induced growth of A549 (lung cancer) and JygM(A) (triple negative breast cancer cell lines) to statistically significant levels. Athymic nude mice were injected with JygM(A) breast cancer cell lines under the mammary fat pads. Mice with palpable tumors were randomized and treated with 3 ug rhTIMP‐2/day/mouse intra‐peritonealy up to a period of 35 days that lead to ~30% reduction of tumor volume compared to the HBSS (vehicle control). We have also evaluated the effects of encapsulation and delivery of rhTIMP‐2 from cationic peptide hydrogel–which shows extended release to up to 28 days with little or no loss of its biologic activity. Similarly, little or no loss of biological activity was observed upon desferoxamine(des) conjugation to rhTIMP‐2. Zn89 attachment to the des subunit has been completed and the Zn89‐rhTIMP‐2 is being used for uptake, distribution and metabolism studies.SUMMARY AND FUTURE DIRECTIONSConclusively we have developed a robust, scalable bioprocess method for rhTIMP‐2 (and analogs) production, characterization to support the availability and analysis through pre‐clinical development. In‐vitro and in‐vivo results shows reduction in cell proliferation and tumor volume in breast, lung cancer cell lines and tumors. Extended release dosage forms and Zn89 labelled TIMP‐2 are now being used to characterize the PK‐PD and distribution of the biologic. With all supportive data, reagents and in‐depth biophysical analysis we present a comprehensive report of rhTIMP‐2 as a novel biologic therapeutic for cancer treatment and we plan to move forward towards continued pre‐clinical/clinical development and protein engineering.Support or Funding InformationThis work is supported by center for cancer research, intramural research program, NCI/NIH research grants ZIA BC011204 &amp; ZIA SC 009179 to WGSS

Inhibition of Eurygaster Integriceps Puton Prolyl Endoprotease (spPEP) and Human Prolyl Endopeptidase (hPEP) using αS1 ‐ Casein Peptide Inhibitors

Prolyl endopeptidases (PEPs) are widely distributed among all domains of life (Di Cera., 2009). The focus of this study is mainly on prolyl endoprotease originated from the salivary glands of Erygaster integriceps Puton (Sunn pest) identified as spPEP by Darkoh (2008, 2010). The widely spread Eurygaster genus represents the most damaging and highly adaptable wheat pest in Northern Africa, Eastern Europe, Middle East with increasing concerns in Australia and United States. The natural substrate for spPEP is gluten from wheat grains, making spPEP the first known eukaryotic PEP capable of degrading substrates longer than 30 amino acid residues. To study the binding mechanism of this unique enzyme, recombinant peptide inhibitors were generated from αS1‐casein containing the inhibitory sequence LNENLLRFFVAPFPEV (Juillerat‐Jeanneret et al., 2011) varying in the length of the peptide and the positioning of the inhibitory sequence. All peptides were constructed with an N‐terminal 6x His Tag for purification as well as a Thrombin protease site for His Tag cleavage, ligated into pET15b vector and transformed into BL21(DE3)pLysS expression host (Vishram.,2016). The expression and purification of the peptides, including thrombin digestion and HPLC for final purification and quantification are shown. Enzyme kinetics for each of the recombinant inhibitory peptides in the presence of GPpNA as substrate is compared between spPEP and human prolyl endopeptidase (hPEP). This is the first study to show the hPEP to bind a peptide longer than 30 amino acid residues as well as inhibition of spPEP with large peptides.

Type I interferons modulate methotrexate resistance in gestational trophoblastic neoplasia.

Resistance to methotrexate is a leading clinical problem in gestational trophoblastic neoplasia (GTN), but there are limited laboratory models for this condition. We created isogenic trophoblastic cell lines resistant to methotrexate and compared these to the parent cell lines using gene expression microarrays and qRT-PCR followed by mechanistic studies using recombinant cytokines, pathway inhibitors, and patient sera. Gene expression microarrays and focused analysis by qRT-PCR revealed methotrexate led to type I interferon upregulation, in particular interferon alpha 2 (IFNA2), and methotrexate resistance was associated with chronic low level increases in type I interferon expression. Recombinant IFNA2 imparted chemosensitive choriocarcinoma cells with partial resistance to methotrexate, while chemoresistant choriocarcinoma cells were uniquely sensitive to fludarabine, a STAT1 inhibitor. In pre-treatment patient sera, IFNA2 levels correlated with subsequent resistance to methotrexate chemotherapy. Methotrexate resistance is influenced by type I interferon signaling with prognostic and therapeutic implications for treating women with GTN.

Sustained response of recombinant human C1 esterase inhibitor for acute treatment of hereditary angioedema attacks

BackgroundSymptoms of hereditary angioedema (HAE) attacks can recur soon after initial treatment; the durability of response for recombinant human C1 esterase inhibitor (rhC1INH) treatment is unknown. ObjectiveTo examine the efficacy and durability of rhC1INH for acute HAE attacks. MethodsIn this pooled post hoc analysis of 2 trials, patients with type I or II HAE (functional C1INH levels <50% of normal) and a baseline visual analog scale score of at least 50 mm were included if they had received at least 1 intravenous dose of 50 IU/kg of rhC1INH. Response was defined as symptom relief within 4 hours after treatment with persistence (≥20-mm decrease in visual analog scale scores [0 mm {“no symptoms at all”} to 100 mm {“extremely disabling”}] at 2 consecutive time points) during the 4 hours. Durability was the response without an increase of at least 20 mm in the minimum post-treatment visual analog scale score up to 24 hours. Recurrence and new attack symptoms were determined for patients with 72-hour post-treatment data. ResultsData were analyzed for 127 patients treated with 50 IU/kg of rhC1INH in 2 studies. Most attacks (90.7%) responded within 4 hours, with differences in response rates among attack locations (61.5%–94.4%). The median time to the beginning of symptom relief was 75.0 minutes (95% confidence interval 65.0–80.0). No relapse occurred during 24 hours for attacks that initially responded. Only 7.1% of attacks were associated with symptom recurrence within 72 hours of initial rhC1INH treatment. ConclusionThis integrated analysis supports the efficacy of rhC1INH for treatment of acute HAE across multiple attacks, with a sustained response for at least 3 days. Trial RegistrationClinicalTrials.gov Identifiers: NCT00225147 and NCT00262301.

Recombinant Human C1-Esterase Inhibitor to Treat Acute Hereditary Angioedema Attacks in Adolescents

Recombinant human C1-esterase inhibitor (rhC1-INH) is efficacious and well tolerated for managing hereditary angioedema (HAE) attacks in adults. However, there are insufficient data on its efficacy and safety in adolescents. To evaluate the efficacy and safety profiles of rhC1-INH for acute HAE attacks in adolescents. Adolescents (aged 12-18 y) with HAE enrolled in 2 randomized controlled trials and 2 open-label extension trials were included and received intravenous rhC1-INH for acute attacks. Times to the beginning of sustained symptom relief (visual analog scale change from baseline ≥20 mm) and minimal symptoms (visual analog scale score of <20 mm across locations) were assessed. Safety parameters included hypersensitivity reactions, anti-rhC1-INH antibodies, and host-related impurities. Sixteen adolescents (50 attacks, aged 14-18 y) received rhC1-INH. Attacks were managed with single-dose rhC1-INH 50 U/kg (46.0%) and single-dose rhC1-INH 2100 U (16%), and 32.0% were treated with additional doses after receiving an initial rhC1-INH 2100 U dose (total dose, 4200-6300 U). Most attacks (88.0%) occurred at a single location; 59.1% (26 of 44) were abdominal. Across the first 5 attacks, median times to the beginning of symptom relief ranged from 19.0 to 78.5 minutes; median times to minimal symptoms ranged from 120 to 190 minutes. Pharmacokinetics showed that rhC1-INH restored functional plasma C1-esterase inhibitor levels to the normal (>70%) range for almost all evaluable patients. No severe or drug-related adverse events or hypersensitivity reactions occurred. No treatment-emergent antibodies to rhC1-INH or host-related impurities were observed. rhC1-INH is efficacious and well tolerated among adolescents with HAE.

Recombinant Human C1 Inhibitor (rHC1INH) Is Efficacious and Well Tolerated As Prophylaxis for Prevention of Hereditary Angioedema (HAE) Attacks: A Randomized, Phase 2 Trial

rhC1INH is currently administered for the treatment of acute HAE attacks. Repeat rhC1INH treatment for recurrent attacks during open-label extension studies have maintained efficacy while being well tolerated, supporting a potential role for rhC1INH in prophylaxis. A randomized, double-blind, 3-period, crossover study was conducted in patients ≥13 years of age with functional C1INH levels <50% of normal and ≥4 HAE attacks during the previous 3 months. Patients received rhC1INH 50 IU/kg (max, 4200 IU) once or twice weekly or placebo in three, 4-week periods; each treatment was separated by 1 week and symptoms monitored by a daily diary. Primary endpoint was number of HAE attacks per 4-week period. Additional endpoints included percentage of patients with ≥25%, ≥50% (clinical response), or ≥75% reduction in number of HAE attacks versus placebo and number of days with HAE symptoms. Thirty-two patients were randomized (mean age, 45.9 years [range, 16.9–73.5 years]). Mean number of HAE attacks was reduced from 7.2 (placebo) to 4.4 (rhC1INH once weekly; P=0.0004) and 2.7 (rhC1INH twice weekly; P<0.0001). Percentage of patients with ≥25%, ≥50%, and ≥75% reduction in HAE attacks versus placebo was 64.5%, 41.9%, and 19.4% for rhC1INH once weekly and 80.6%, 74.2%, and 41.9% rhC1INH twice weekly, respectively. Number of days with HAE symptoms was lower with rhC1INH twice weekly (5.1 days) and once weekly (8.0 days) versus placebo (10.2 days). rhC1INH was efficacious for the prevention of HAE attacks and data support continued investigation of rhC1INH as prophylaxis.

RBTI reduced β-amyloid-induced toxicity by promoting autophagy-lysosomal degradation via DAF-16 in Caenorhabditis elegans

Alzheimer's disease (AD) is an age-related neurodegenerative disease, of which β-amyloid (Aβ) induced toxicity was suggested as a main cause. Some substances with prolongevity effects have been shown to be protective against AD. In a previous study we demonstrated that a recombinant buckwheat trypsin inhibitor (rBTI) could prolonge the lifespan in Caenorhabditis elegans (C. elegans). Here, we investigated whether rBTI may benefit to mitigate the AD symptom by feeding the AD model C. elegans CL4176. CL4176 is a transgenic C. elegans expressing human Aβ3–42 in muscle tissue. The results showed that rBTI not only could extend lifespan but also could reduce Aβ toxicity-triggered body paralysis in AD worms. Further study found the accumulation of Aβ was decreased and autophagy-lysosomal degradation pathway was activated in AD worms treated with rBTI. Moreover, the inhibition of autophagy reduced rBTI-mediated paralysis delay. Genetic analyses showed rBTI increased the transcriptional activity of dauer formation abnormal-16 (DAF-16) and the disruption of daf-16 abolished rBTI-mediated protective effect in AD worms. Taken together, these data indicated that rBTI promoted the autophagy-lysosomal degradation pathway to reduce the Aβ-induced toxicity via DAF-16 in an AD model C. elegans, implying that BTI has the potential to protect against AD.

Plasma kallikrein enhances platelet aggregation response by subthreshold doses of ADP

Human plasma kallikrein (huPK) potentiates platelet responses to subthreshold doses of ADP, although huPK itself, does not induce platelet aggregation. In the present investigation, we observe that huPK pretreatment of platelets potentiates ADP-induced platelet activation by prior proteolysis of the G-protein-coupled receptor PAR-1. The potentiation of ADP-induced platelet activation by huPK is mediated by the integrin αIIbβ3 through interactions with the KGD/KGE sequence motif in huPK. Integrin αIIbβ3 is a cofactor for huPK binding to platelets to support PAR-1 hydrolysis that contributes to activation of the ADP signaling pathway. This activation pathway leads to phosphorylation of Src, AktS473, ERK1/2, and p38 MAPK, and to Ca2+ release. The effect of huPK is blocked by specific antagonists of PAR-1 (SCH 19197) and αIIbβ3 (abciximab) and by synthetic peptides comprising the KGD and KGE sequence motifs of huPK. Further, recombinant plasma kallikrein inhibitor, rBbKI, also blocks this entire mechanism. These results suggest a new function for huPK. Formation of plasma kallikrein lowers the threshold for ADP-induced platelet activation. The present observations are consistent with the notion that plasma kallikrein promotes vascular disease and thrombosis in the intravascular compartment and its inhibition may ameliorate cardiovascular disease and thrombosis.

Recombinant hepatocyte growth factor activator inhibitor 1: expression in Drosophila S2 cells, purification and crystallization.

Hepatocyte growth factor activator inhibitor 1 (HAI-1) is a multi-domain membrane-associated protease inhibitor that potently inhibits a variety of serine proteases such as hepatocyte growth factor activator and matriptase. Different truncates of HAI-1 show varying potencies for inhibition of target proteases, suggesting that the domain organization of HAI-1 plays a critical role in its function. Here, the soluble full-length extracellular part of HAI-1 (sHAI-1) was expressed using the Drosophila S2 insect-cell expression system. Diffraction-quality crystals of sHAI-1 were produced using ammonium sulfate as precipitant. The crystal diffracted to 3.8 Å resolution and belonged to space group P41212, with unit-cell parameters a = b = 95.42, c = 124.50 Å. The asymmetric unit contains one sHAI-1 molecule.

Capillaries, Old Age and Alzheimer’s Disease

Many of the minor complaints of old age may have a common etiology and are grouped together here under the term ‘the lesser ailments of aging’ (LAA). This essay proposes that they are due in large part to an age-linked reduced microcirculation. Capillary density (CD) in the tissues is determined by levels of angiogenic growth factors (AGFs). Over 47 studies have reported a reduced CD and/or waning AGFs throughout the bodies of aging animals and people. More convincing than such a generalization are the 80 sets of data comparing these two parameters in adult vs. the aged. These data have led to a hypothesis whose corollary proposes a specific treatment for the LAA. While genetically controlled, the waning levels of AGFs theoretically could be countered by pro-angiogenesis therapy and thus might ease the LAA or delay their onset. Therapies mentioned here include recombinant AGFs and inhibitors of type 5 phosphodiesterases, such a tadalafil/Cialis. Finally, Alzheimer’s disease (AD) is generally an illness of the elderly and may have a single or multiple causes. However, its clinical course may be influenced secondarily by conditions affecting the LAA. Therefore, any effective treatment of them may influence favorably the clinical course of AD.

O001 Randomized, double-blind, placebo-controlled trial of recombinant human C1 inhibitor for prophylaxis of hereditary angioedema attacks

O001 Randomized, double-blind, placebo-controlled trial of recombinant human C1 inhibitor for prophylaxis of hereditary angioedema attacks