Kallikrein Activity Research Articles

Ulinastatin attenuated cardiac ischaemia/reperfusion injury by suppressing activation of the tissue kallikrein-kinin system.

Ulinastatin has beneficial effects in patients undergoing coronary artery bypass grafting (CABG) surgery due to its anti-inflammatory properties, but the underlying mechanism remains unclear. We used samples from patients undergoing CABG, a model of cardiac ischaemia-reperfusion injury (IRI) in mice and murine cardiac endothelial cell cultures to investigate links between ulinastatin, the kallikrein-kinin system (KKS), endothelial dysfunction and cardiac inflammation in the response to ischaemia/reperfusion injury (IRI). These links were assessed using clinical investigations, in vitro and in vivo experiments and RNA sequencing analysis. Ulinastatin inhibited the activity of tissue kallikrein, a key enzyme of the KKS, at 24 h after CABG surgery, which was verified in our murine cardiac ischaemia-reperfusion model. Under normal conditions, ulinastatin only inhibited kallikrein activity but did not affect bradykinin (B1/B2) receptors. Ulinastatin protected against IRI, in vivo and in vitro, by suppressing activation of the kallikrein-kinin system and down-regulating B1/B2 receptor-related signalling pathways including ERK/ iNOS, which resulted in enhanced endothelial barrier function, mitigation of inflammation and oedema, decreased infarct size, improved cardiac function and decreased mortality. Inhibition of kallikrein and knockdown of B1, but not B2 receptors prevented ERK translocation into the nucleus, reducing reperfusion-induced injury in murine cardiac endothelial cells. Treatment with ulinastatin exerts a protective influence on cardiac reperfusion by suppressing activation of the kallikrein-kinin system. Our findings highlight the potential of targeting kallikrein /bradykinin receptors to alleviate endothelial dysfunction, thus improving cardiac IRI.

Abstract P199: Renal kallikrein-1 is not essential in adaptation to an overnight potassium load

Background: Kallikrein-1 is a serine protease expressed in multiple tissues including the kidney, where it is secreted into the urine. Low urinary kallikrein levels are associated with essential hypertension, whereas high levels are associated with primary hyperaldosteronism in humans. Global kallikrein-1 deletion in mice has been shown to lead to hyperkalemia in response to an acute potassium load as well as defective cleavage of gamma-ENaC, suggesting a direct role in distal K + handling. However, it remains unclear whether effects seen with global kallikrein-1 loss are secondary to loss of extrarenal or renal kallikrein, which is primarily expressed in the connecting tubule. Methods: To test the hypothesis that renal kallikrein-1 is necessary for increased K + secretion in response to acute dietary K + load, we generated mice with conditional knockout of kallikrein-1 in the distal tubule (Klk1 flox - Calbindin-Cre, KS-Klk1 KO). Mice were treated with a K + deficient diet (0% K + , LK) for 9.5 days and then placed in metabolic cages for 12hr overnight collection of urine on control (LK) or high K + (5%, OHK) diets. Metabolic parameters in plasma were measured using iSTAT and kidneys were harvested for downstream western blotting. Results: Deletion of kallikrein-1 in KS-Klk1 kidney was confirmed by western blotting and urine kallikrein activity assay. On LK diet, both WT and KS-Klk1 KO mice developed marked hypokalemia (WT 2.83+/-0.23 vs KO 2.88+/-0.15 meq/dL). Following OHK challenge, hyperkalemia developed to similar levels in both groups (WT 7.13+/-0.56 vs KO 7.74+/-0.35). However, KS-Klk1 KO mice with HK challenge exhibited a more pronounced drop in serum [Na + ] compared with WT (WT 142.6+/-1.4 vs KO 136.6+/-1.3, p=0.022) and correspondingly lower serum [Cl - ] (WT 119.0+/-0.62 vs KO 115.9+/-0.96). Gamma-ENaC quantitation by western blot revealed similar ratios of cleaved: uncleaved species between genotypes. No significant changes between genotypes were detected in urine volume or urine Na+/K+ ratios. Conclusions: KS-Klk1 KO mice do not display defective K+ handling or ENaC processing in response to an acute 12 hr overnight high K + diet. However, they do display relative hyponatremia compared with WT. These data may suggest compensatory mechanisms for potassium transport or a direct a role for renal kallikrein-1 in regulation of renal water transport.

Effect of Various Fruit Extracts on Angiotensin I-Converting Enzyme (ACE) and Kallikrein (KLK) Activities.

Hypertension is one of the main risk factors for cardiovascular disease and causes widespread morbidity and mortality worldwide. The aim of this work was to screen the fruit with high angiotensin I-converting enzyme (ACE) inhibitory activity and kallikrein (KLK) promotion activity by three different extraction methods from 22 kinds of fruits. Results showed that the aqueous extracts of fresh kiwifruit significantly inhibited ACE activity (47.71%), whereas the KLK activity was also inhibited (4.56%). This indicated that the substances inhibiting ACE activity existed in kiwifruit might be small molecular substances such as polyphenols. The nonpolar substance existed in the ethanol extracts of grape inhibited ACE activity significantly. The enzymatic hydrolysates of red grape significantly promoted KLK activity, whereas its ethanol extracts significantly inhibited KLK activity. This results suggested that the components that lower blood pressure and raise blood pressure are generally presented in the same fruit, the former are mostly water-soluble substances, while the latter are generally alcohol-soluble substances. If certain or individual components can be isolated from edible fruits, they may significantly affect blood pressure in humans.

A quantitative systems pharmacology model of plasma kallikrein-kinin system dysregulation in hereditary angioedema.

Hereditary angioedema (HAE) due to C1-inhibitor deficiency is a rare, debilitating, genetic disorder characterized by recurrent, unpredictable, attacks of edema. The clinical symptoms of HAE arise from excess bradykinin generation due to dysregulation of the plasma kallikrein-kinin system (KKS). A quantitative systems pharmacology (QSP) model that mechanistically describes the KKS and its role in HAE pathophysiology was developed based on HAE attacks being triggered by autoactivation of factor XII (FXII) to activated FXII (FXIIa), resulting in kallikrein production from prekallikrein. A base pharmacodynamic model was constructed and parameterized from literature data and ex vivo assays measuring inhibition of kallikrein activity in plasma of HAE patients or healthy volunteers who received lanadelumab. HAE attacks were simulated using a virtual patient population, with attacks recorded when systemic bradykinin levels exceeded 20pM. The model was validated by comparing the simulations to observations from lanadelumab and plasma-derived C1-inhibitor clinical trials. The model was then applied to analyze the impact of nonadherence to a daily oral preventive therapy; simulations showed a correlation between the number of missed doses per month and reduced drug effectiveness. The impact of reducing lanadelumab dosing frequency from 300mg every 2weeks (Q2W) to every 4weeks (Q4W) was also examined and showed that while attack rates with Q4W dosing were substantially reduced, the extent of reduction was greater with Q2W dosing. Overall, the QSP model showed good agreement with clinical data and could be used for hypothesis testing and outcome predictions.

Kinin-kallikrein system: New perspectives in heart failure.

Heart failure (HF) is a pervasive clinical challenge characterized by compromised cardiac function and reduced quality of life. The kinin-kallikrein system (KSS), a multifaceted peptide cascade, has garnered substantial attention due to its potential role in HF. Through activation of B1 and/or B2 receptors and downstream signaling, kinins modulate various physiological processes, including inflammation, coagulation, pain, blood pressure control, and vascular permeability. Notably, aberrations in KKS components have been linked to HF risk. The elevation of vasodilatory bradykinin (BK) due to kallikrein activity reduces preload and afterload, while concurrently fostering sodium reabsorption inhibition. However, kallikrein's conversion of prorenin to renin leads to angiotensinsII upregulation, resulting in vasoconstriction and fluid retention, alongside increased immune cell activity that fuels inflammation and cardiac remodeling. Importantly, prolonged KKS activation resulting from volume overload and tissue stretch contributes to cardiac collagen loss. The conventional renin-angiotensin-aldosterone system (RAAS) inhibitors used in HF management may inadvertently intensify KKS activity, exacerbating collagen depletion and cardiac remodeling. It is crucial to balance the KKS's role in acute cardiac damage, which may temporarily enhance function and metabolic parameters against its detrimental long-term effects. Thus, KKS blockade emerges as a promising strategy to impede HF progression. By attenuating the link between immune system function and tissue damage, KKS inhibition can potentially reduce cardiac remodeling and alleviate HF symptoms. However, the nuanced roles of BK in various acute conditions necessitate further investigation into the sustained benefits of kallikrein inhibitors in patients with chronic HF.

Recessive SERPING1 Variant Leads to Kinin–Kallikrein System Control Failure in a Consanguineous Brazilian Family with Hereditary Angioedema

Background: Hereditary angioedema (HAE) is a severe and potentially life-threatening disease. The most common forms are caused by variants in SERPING1, resulting in C1-inhibitor (C1-INH) deficiency (HAE-C1-INH). C1-INH is a serine protease inhibitor (SERPIN) that regulates multiple proteases pathways, including the kallikrein–kinin system (KKS) and its complement. In HAE-C1-INH patients, C1-INH deficiencies affect KKS control, resulting in the development of kallikrein activity in plasma and the subsequent release of bradykinin (BK). While the overwhelming majority of disease-causing SERPING1 variants are dominant, very few recessive variants have been described. We present a large Brazilian HAE-C1-INH family with a recessive form of HAE-C1-INH. Methods: Blood samples of family members were investigated for protein levels of C1-INH, C4, C1q, and C1-INH function. The SERPING1 gene was sequenced. Results: In two severely affected sisters, we identified a homozygous missense variant in SERPING1 (NM_000062.3:c.964G>A;p.Val322Met). Fourteen family members were asymptomatic heterozygous carriers of the variant. Data regarding C1-INH function in the plasma showed that homozygous p.Val322Met strongly impacts C1-INH function to inhibit C1s and kallikrein (PKa). When heterozygously expressed, it affects the C1-INH control of C1s more than that of PKa. Conclusions: These studies of the variant’s effects on the structure–function relationship reinforce prior observations suggesting that C1-INH deficiency is a conformational disease.

Role of Tranexamic acid in NSAIDS induced angioedema

Tranexamic acid (TXA) is an antifibrinolytic agent that inhibits the conversion of plasminogen to plasmin, a key step in kallikrein activation and bradykinin formation. Tranexamic acid is used in the prophylactic management of hereditary angioedema; however, evidence for TXA in Non-Steroidal Anti-inflammatory drug-induced angioedema (NSAIDS-AE) is limited. We describe a patient who presented to the ICU department with NSAIDS-AE and was successfully treated with TXA. This case suggests that TXA may be a beneficial treatment modality in the management of NSAIDS-AE and warrants further investigation.

STAR-0215 is a Novel, Long-Acting Monoclonal Antibody Inhibitor of Plasma Kallikrein for the Potential Treatment of Hereditary Angioedema.

Hereditary angioedema (HAE) is a rare autosomal dominant disorder caused by a deficiency in functional C1 esterase inhibitor (C1-INH), a serpin family protein that blocks the activity of plasma kallikrein. Insufficient inhibition of plasma kallikrein results in the overproduction of bradykinin, a vasoactive inflammatory mediator that produces both pain and unpredictable swelling during HAE attacks, with potentially life-threatening consequences. We describe the generation of STAR-0215, a humanized immunoglobulin G1 (IgG1) antibody with a long circulating half-life (t1/2) that potently inhibits plasma kallikrein activity, with a >1000-fold lower affinity for prekallikrein and no measurable inhibitory activity against other serine proteases. The high specificity and inhibitory effect of STAR-0215 is demonstrated through a unique allosteric mechanism involving N-terminal catalytic domain binding, destabilization of the activation domain, and reversion of the active site to the inactive zymogen state. The YTE (M252Y/S254T/T256E) modified fragment crystallizable (Fc) domain of STAR-0215 enhances pH-dependent neonatal Fc receptor (FcRn) binding, resulting in a prolonged t1/2 in vivo (⁓34 days in cynomolgus monkeys) compared to antibodies without this modification. A single subcutaneous dose of STAR-0215 ({greater than or equal to} 100 mg) was predicted to be active in patients for 3 months or longer, based on simulations using a minimal physiologically based pharmacokinetic (mPBPK) model. These data indicate that STAR-0215, a highly potent and specific antibody against plasma kallikrein with extended t1/2, is a potential agent for long-term preventative HAE therapy administered every 3 months or less frequently. Significance Statement STAR-0215 is a YTE-modified immunoglobulin G1 monoclonal antibody with a novel binding mechanism that specifically and potently inhibits the enzymatic activity of plasma kallikrein and prevents the generation of bradykinin. It has been designed to be a long-lasting prophylactic treatment to prevent attacks of HAE and to decrease the burden of disease and the burden of treatment for people with HAE.

Drugs of the Kallikrein–Kinin System: An Overview

The kallikrein–kinin system consists of the two kininogen substrates present in the blood plasma, and two serine proteases: the plasma and tissue kallikreins. The action of the latter on kininogens produces small peptides, the kinins, short-lived, but endowed by powerful pharmacologic actions on blood vessels and other tissues. Many recent and exciting therapeutic developments in the field are briefly summarized. Notably, various novel strategies are being clinically developed to inhibit the formation of bradykinin or block its receptors in the management of hereditary angioedema. The interventions include orally bioavailable drugs, biotechnological proteins, and gene therapy. These approaches are currently explored in a variety of other inflammatory and thrombotic disorders. Harnessing controlled kinin formation is also of potential therapeutic interest, as shown by the clinical development of recombinant tissue kallikrein for ischemic stroke and renal disease. The biomarkers of kinin-mediated disorders, frequently implicating edemas, include the consumption of kininogen(s), plasma kallikrein activity, and the detection of circulating kinin metabolites such as fragments BK1–5 and BK2–9. Novel opportunities to clinically apply the underexploited drugs of the kallikrein–kinin system are briefly reviewed. This personal perspective is offered by an observer of and a participant in drug characterization throughout the last four decades.

Isolation and Characterization of Poeciguamerin, a Peptide with Dual Analgesic and Anti-Thrombotic Activity from the Poecilobdella manillensis Leech

When Poecilobdella manillensis attacks its prey, the prey bleeds profusely but feels little pain. We and other research teams have identified several anticoagulant molecules in the saliva of P. manillensis, but the substance that produces the paralyzing effect in P. manillensis is not known. In this study, we successfully isolated, purified, and identified a serine protease inhibitor containing an antistasin-like domain from the salivary secretions of P. manillensis. This peptide (named poeciguamerin) significantly inhibited elastase activity and slightly inhibited FXIIa and kallikrein activity, but had no effect on FXa, trypsin, or thrombin activity. Furthermore, poeciguamerin exhibited analgesic activity in the foot-licking and tail-withdrawal mouse models and anticoagulant activity in the FeCl3-induced carotid artery thrombosis mouse model. In this study, poeciguamerin was found to be a promising elastase inhibitor with potent analgesic and antithrombotic activity for the inhibition of pain and thrombosis after surgery or in inflammatory conditions.

Efficacy and Safety of Garadacimab in Combination with Standard of Care Treatment in Patients with Severe COVID-19

BackgroundGaradacimab, a fully human IgG4 monoclonal antibody, inhibits the kallikrein–kinin pathway at a key initiator, activated coagulation factor XII (FXIIa), and may play a protective role in preventing the progression of COVID-19. This phase 2 study evaluated the efficacy and safety of garadacimab plus standard of care (SOC) versus placebo plus SOC in patients with severe COVID-19.MethodsPatients hospitalised with COVID-19 were randomised (1:1) to a single intravenous dose of garadacimab (700 mg) plus SOC or placebo plus SOC. Co-primary endpoint was incidence of endotracheal intubation or death between randomisation and Day 28. All-cause mortality, safety and pharmacokinetic/pharmacodynamic parameters were assessed.ResultsNo difference in incidence of tracheal intubation or death (p = 0.274) or all-cause mortality was observed (p = 0.382). Garadacimab was associated with a lower incidence of treatment-emergent adverse events (60.3% vs 67.8%) and fewer serious adverse events (34 vs 45 events) versus placebo. No garadacimab-related deaths or bleeding events were reported, including in the 45.9% (n = 28/61) of patients who received concomitant heparin. Prolonged activated partial thromboplastin time (aPTT), and increased coagulation factor XII (FXII) levels were observed with garadacimab versus placebo to Day 14, whilst FXIIa-mediated kallikrein activity (FXIIa-mKA) was suppressed to Day 28.ConclusionIn patients with severe COVID-19, garadacimab did not confer a clinical benefit over placebo. Transient aPTT prolongation and suppressed FXIIa-mKA showed target engagement of garadacimab that was not associated with bleeding events even with concomitant anticoagulant use. The safety profile of garadacimab was consistent with previous studies in patients with hereditary angioedema.ClinicalTrials. gov IdentifierNCT04409509. Date of registration: 28 May, 2020.

Predictive value for increased activated factor XI activity in acute venous thromboembolism

BackgroundVenous thromboembolism (VTE) is associated with excessive coagulation activity, which in part can be attributed to activation of contact system. However, the knowledge regarding the impact of contact activation in acute VTE is limited. ObjectiveTo unravel the involvement of contact activation in acute VTE. MethodsContact activation was investigated in patients with acute VTE (n = 321) and population controls without a history of VTE (n = 300). For comparison, Factor XI(a) levels, activity, and plasma kallikrein (PKa) activity were determined in plasma samples with an activated partial thromboplastin time– or thrombin generation-based assay (free FXI concentration [FXI:c] and calibrated automated thrombogram:FXIa, respectively) and with enzyme-linked immunosorbent assays for enzyme-inhibitor complexes (FXIa:alpha-1-antitrypsin [α1AT], FXIa:antithrombin [AT], FXIa:C1-inhibitor [C1Inh], and PKa:C1-inh). ResultsIn patients with VTE, higher FXI:c levels (124 ± 37% vs 114 ± 28%), but lower calibrated automated thrombogram:FXIa levels were apparent. This was accompanied by increased FXIa:α1AT, FXIa:AT, and PKa:C1-inh levels in patients compared with controls (312pM [238–424] vs 203pM [144–288]; 29pM [23–38] vs 23pM [20–30]; 1.9nM [1.2–4.7] vs 1.4nM [0.7–3.5], respectively), whereas FXIa:C1-inh levels did not differ. Logistic regression models showed good discriminatory values for FXI:c and FXIa:α1AT (area under the curve = 0.64 [0.6/0.69] and 0.73 [0.69/0.77], respectively). After a 2-year follow-up, 81 recurrent VTE events or deaths occurred in the patient cohort, for which the baseline levels of FXIa:α1AT and FXIa:C1Inh had a significant prognostic value (Hazard ratios per SD [95% CI], 1.26 [1.10–1.45]; p =.0012 and 1.19 [1.05–1.36]; p =.0082, respectively). ConclusionOur study revealed elevated FXIa levels and activity in acute VTE, which was also associated with recurrent VTE, suggesting an important risk contribution of FXI activation to VTE. The evidence provided by this study supports the utility of FXIa inhibition in the setting of acute VTE.

Moisturizing at a molecular level - The basis of Corneocare.

This review covers the last 20 years of research we and our collaborators have conducted on ethnic differences in facial skin moisturization placed in historical context with previous research. We have focussed particularly on the biochemical and cellular gradients of the stratum corneum (SC) with the aim of discovering new skin moisturization and SC maturation mechanisms, identifying new technologies and/or providing conceptual innovations for ingredients that will improve our understanding and treatment of dry skin. Specifically, we discuss gradients for corneodesmosomes and proteases, corneocyte phenotype-inducing enzymes, filaggrin and natural moisturizing factor (NMF), and barrier lipids. These gradients are interdependent and influence greatly corneocyte maturation. The interrelationship between corneodesmolysis and the covalent attachment of ω-hydroxy ceramides and ω-hydroxy fatty acids to the corneocyte protein envelope forming the corneocyte lipid envelope is especially relevant in our new understanding of mechanisms leading to dry skin. This process is initiated by a linoleoyl-ω-acyl ceramide transforming enzyme cascade including 12R lipoxygenase (12R-LOX), epidermal lipoxygenase-3 (eLOX3), epoxide hydrolase 3 (EPHX3), short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7), ceramidase and transglutaminase 1. Our research has opened the opportunity of using novel treatment systems for dry skin based on lipids, humectants, niacinamide and inhibitors of the plasminogen system. It is clear that skin moisturization is a more complex mechanism than simple skin hydration.

Type I IFNs link skin-associated dysbiotic commensal bacteria to pathogenic inflammation and angiogenesis in rosacea.

Rosacea is a common chronic inflammatory skin disease with a fluctuating course of excessive inflammation and apparent neovascularization. Microbial dysbiosis with a high density of Bacillus oleronius and increased activity of kallikrein 5, which cleaves cathelicidin antimicrobial peptide, are key pathogenic triggers in rosacea. However, how these events are linked to the disease remains unknown. Here, we show that type I IFNs produced by plasmacytoid DCs represent the pivotal link between dysbiosis, the aberrant immune response, and neovascularization. Compared with other commensal bacteria, B. oleronius is highly susceptible and preferentially killed by cathelicidin antimicrobial peptides, leading to enhanced generation of complexes with bacterial DNA. These bacterial DNA complexes but not DNA complexes derived from host cells are required for cathelicidin-induced activation of plasmacytoid DCs and type I IFN production. Moreover, kallikrein 5 cleaves cathelicidin into peptides with heightened DNA binding and type I IFN-inducing capacities. In turn, excessive type I IFN expression drives neoangiogenesis via IL-22 induction and upregulation of the IL-22 receptor on endothelial cells. These findings unravel a potentially novel pathomechanism that directly links hallmarks of rosacea to the killing of dysbiotic commensal bacteria with induction of a pathogenic type I IFN-driven and IL-22-mediated angiogenesis.

Initial Results from a Phase 1 Single Ascending Dose Clinical Trial of STAR-0215, an Investigational Long-Acting Monoclonal Antibody Plasma Kallikrein Inhibitor for Hereditary Angioedema (HAE), in Healthy Subjects Followed for at Least 3 Months

The majority of HAE cases are caused by C1-INH deficiencies, resulting in uncontrolled activation of plasma kallikrein. STAR-0215 is an investigational humanized IgG1kappa monoclonal antibody with potent and durable (≥3 month) reduction of plasma kallikrein activity demonstrated in cynomolgus monkeys. This Phase 1 trial (NCT05477160) is assessing safety and the pharmacokinetic (PK) and pharmacodynamic (PD) activities that support subcutaneous administration of STAR-0215 to achieve potentially robust and durable suppression of HAE attacks for 3 months or longer.

Structure of STAR-0215 Bound to Active Plasma Kallikrein Reveals a Novel Mechanism of Enzyme Inhibition

Inhibition of plasma kallikrein (pKal) activity is a validated mechanism for prevention of hereditary angioedema (HAE) attacks. High potency and long duration of action are key drivers of prophylactic efficacy of pKal inhibitors. STAR-0215 is a novel, potent and selective long-acting monoclonal antibody plasma kallikrein pKal inhibitor in clinical development. In monkeys dosed subcutaneously, STAR-0215 inhibits pKal activity rapidly and durably. We investigated STAR-0215 structure and binding to pKal and its inhibitory mechanism of action by utilizing Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) and Cryogenic Electron Microscopy (Cryo-EM).

New medications to mitigate attacks of hereditary angioedema: does one size fit all?

Hereditary angioedema has received renewed attention in the past two decades, with the elucidation of the biochemical pathways resulting in bradykinin overproduction in patients with congenital C1esterase inhibitor (C1INH) deficiency, leading to recurrent attacks of tissue swelling due to uncontrolled vascular permeability. 1 Busse PJ Christiansen SC Hereditary angioedema. N Engl J Med. 2020; 382: 1136-1148 Crossref PubMed Scopus (132) Google Scholar Oedema attacks affecting the lingual or laryngeal area can become lethal as a result of asphyxiation. 2 Minafra FG Gonçalves TR Alves TM Pinto JA The mortality from hereditary angioedema worldwide: a review of the real-world data literature. Clin Rev Allergy Immunol. 2022; 62: 232-239 Crossref PubMed Scopus (4) Google Scholar Several new treatments have emerged, 143 years after Quincke's first description of hereditary angioedema and 15 years after the introduction of the first specific bradykinin B2 receptor inhibitor (icatibant). These drugs specifically target crucial steps in the kallikrein–kinin (contact) cascade downstream of bradykinin production. 3 Fijen LM Bork K Cohn DM Current and prospective targets of pharmacologic treatment of hereditary angioedema types 1 and 2. Clin Rev Allergy Immunol. 2021; 61: 66-76 Crossref PubMed Scopus (0) Google Scholar Many of these agents have shown their efficacy and safety in acute (on-demand) treatment and prophylaxis of hereditary angioedema attacks, saving lives and revolutionising patients’ quality of life. 4 Lumry WR Settipane RA Hereditary angioedema: epidemiology and burden of disease. Allergy Asthma Proc. 2020; 41: S08-S Crossref Google Scholar These modalities fit well into the modern paradigm in hereditary angioedema treatment, supporting patients’ autonomy, recommending self-administration, and reducing reliance on hospital emergency rooms, health-care providers, and medical points of care. 5 Maurer M Magerl M Betschel S et al. The international WAO/EAACI guideline for the management of hereditary angioedema—the 2021 revision and update. Allergy. 2022; 77: 1961-1990 Crossref PubMed Scopus (32) Google Scholar An investigational oral plasma kallikrein inhibitor for on-demand treatment of hereditary angioedema: a two-part, randomised, double-blind, placebo-controlled, crossover phase 2 trialOral administration of sebetralstat was well tolerated and led to rapid suppression of plasma kallikrein activity, resulting in increased time to use of conventional attack treatment and faster symptom relief versus placebo. Based on these results, a phase 3 trial to evaluate the efficacy and safety of two dose levels of sebetralstat in adolescent and adult participants with hereditary angioedema has been initiated ( NCT05259917 ). Full-Text PDF

An investigational oral plasma kallikrein inhibitor for on-demand treatment of hereditary angioedema: a two-part, randomised, double-blind, placebo-controlled, crossover phase 2 trial

Guidelines recommend effective on-demand therapy for all individuals with hereditary angioedema. We aimed to assess the novel oral plasma kallikrein inhibitor, sebetralstat, which is in development, for on-demand treatment of hereditary angioedema attacks. In this two-part phase 2 trial, individuals with type 1 or 2 hereditary angioedema aged 18 years or older were recruited from 25 sites, consisting of specialty outpatient centres, across nine countries in Europe and the USA. Individuals were eligible if they had experienced at least three hereditary angioedema attacks in the past 93 days, were not on prophylactic therapy, and had access to and the ability to self-administer conventional attack treatment. In part 1 of the trial, participants were given a single 600 mg open-label oral dose of sebetralstat to assess safety, pharmacokinetics, and pharmacodynamics of the dose. Part 2 was a randomised, double-blind, placebo-controlled, two-sequence, two-period (2 × 2) crossover trial; participants were randomly assigned (1:1) to either sequence 1, in which they were given a single dose of 600 mg of sebetralstat to treat the first eligible attack and a second dose of placebo to treat the second eligible attack, or sequence 2, in which they were given placebo to treat the first eligible attack and then 600 mg of sebetralstat to treat the second eligible attack. Participants and investigators were masked to treatment assignment. The primary endpoint was time to use of conventional attack treatment within 12 h of study drug administration, which was assessed in all participants who were randomly assigned to treatment and who received study drug for two attacks during part 2 of the study. Safety was assessed in all participants who received at least one dose of study drug, starting in part 1. This study is registered with ClinicalTrials.gov, NCT04208412, and is completed. Between July 2, 2019, and Dec 8, 2020, 84 individuals were screened and 68 were enrolled in part 1 and received sebetralstat (mean age 38·3 years [SD 13·2], 37 [54%] were female, 31 [46%] were male, 68 [100%] were White). 42 (62%) of 68 participants completed pharmacokinetic assessments. Sebetralstat was rapidly absorbed, with a geometric mean plasma concentration of 501 ng/mL at 15 min. In a subset of participants (n=6), plasma samples obtained from 15 min to 4 h after study drug administration had near-complete protection from ex vivo stimulated generation of plasma kallikrein and cleavage of high-molecular-weight kininogen. In part 2, all 68 participants were randomly assigned to sequence 1 (n=34) or sequence 2 (n=34). 53 (78%) of 68 participants treated two attacks (25 [74%] in the sequence 1 group and 28 [82%] in the sequence 2 group). Time to use of conventional treatment within 12 h of study drug administration was significantly longer with sebetralstat versus placebo (at quartile 1: >12 h [95% CI 9·6 to >12] vs 8·0 h [3·8 to >12]; p=0·0010). There were no serious adverse events or adverse event-related discontinuations. Oral administration of sebetralstat was well tolerated and led to rapid suppression of plasma kallikrein activity, resulting in increased time to use of conventional attack treatment and faster symptom relief versus placebo. Based on these results, a phase 3 trial to evaluate the efficacy and safety of two dose levels of sebetralstat in adolescent and adult participants with hereditary angioedema has been initiated (NCT05259917). KalVista Pharmaceuticals.

Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin.

Protection from the toxicity of nerve agents is achieved by pretreatment with human butyrylcholinesterase (BChE). Current methods for purifying large quantities of BChE from frozen Cohn fraction IV-4 produce 99% pure enzyme, but the yield is low (21%). Our goal was to simplify the purification procedure and increase the yield. Butyrylcholinesterase was extracted from frozen Cohn fraction IV-4 in 10 volumes of water pH 6. The filtered extract was pumped onto a Hupresin affinity column. The previously utilized anion exchange chromatography step was omitted. Solvent and detergent reagents used to inactivate lipid enveloped virus, bacteria and protozoa did not bind to Hupresin. BChE was eluted with 0.1 M tetramethylammonium bromide in 20 mM sodium phosphate pH 8.0. BChE protein was concentrated on a Pellicon tangential flow filtration system and demonstrated to be highly purified by mass spectrometry. A high pump rate produced protein aggregates, but a low pump rate caused minimal turbidity. Possible contamination by prekallikrein and prekallikrein activator was examined by LC-MS/MS and by a chromogenic substrate assay for kallikrein activity. Prekallikrein and kallikrein were not detected by mass spectrometry in the 99% pure BChE. The chromogenic assay indicated kallikrein activity was less than 9 mU/mL. This new, 1-step chromatography protocol on Hupresin increased the yield of butyrylcholinesterase by 200%. The new method significantly reduces production costs by optimizing yield of 99% pure butyrylcholinesterase.

Mesenchymal stem cells express epidermal markers in an in vitro reconstructed human skin model.

Introduction: In skin traumas, such as burns, epidermal homeostasis is affected, often requiring clinical approaches. Different therapeutic strategies can be used including transplantation, besides the use of synthetic or natural materials with allogeneic cells. In this context, tissue engineering is an essential tool for skin regeneration, and using mesenchymal stem cells (MSC) from the umbilical cord appears to be a promising strategy in regenerative medicine due to its renewal and differentiation potential and hypo immunogenicity. We evaluated the transdifferentiation of MSC from umbilical cord into keratinocytes in three-dimensional (3D) in vitro skin models, using dermal equivalents composed by type I collagen with dermal fibroblasts and a commercial porcine skin decellularized matrix, both cultured at air-liquid interface (ALI). Methods: The expression of epidermal proteins cytokeratins (CK) 5, 14 and 10, involucrin and filaggrin was investigated by real-time PCR and immunofluorescence, in addition to the activity of epidermal kallikreins (KLK) on the hydrolysis of fluorogenic substrates. Results and discussion: The cultivation of MSCs with differentiation medium on these dermal supports resulted in organotypic cultures characterized by the expression of the epidermal markers CK5, CK14, CK10 and involucrin, mainly on the 7th day of culture, and filaggrin at 10th day in ALI. Also, there was a 3-fold increase in the KLK activity in the epidermal equivalents composed by MSC induced to differentiate into keratinocytes compared to the control (MSC cultivated in the proliferation medium). Specifically, the use of collagen and fibroblasts resulted in a more organized MSC-based organotypic culture in comparison to the decellularized matrix. Despite the non-typical epithelium structure formed by MSC onto dermal equivalents, the expression of important epidermal markers in addition to the paracrine effects of these cells in skin may indicate its potential use to produce skin-based substitutes.