The effect of cumulating exposure to abacavir on the risk of cardiovascular disease events in patients from the Swiss HIV Cohort Study.

BACKGROUND Patients with HIV exposed to the antiretroviral drug abacavir may have an increased risk of cardiovascular disease (CVD). There is concern that this association arises because of a channelling bias. Even if exposure is a risk, it is not clear how that risk changes as exposure cumulates. METHODS We assess the effect of exposure to abacavir on the risk of CVD events in the Swiss HIV Cohort Study. We use a new marginal structural Cox model to estimate the effect of abacavir as a flexible function of past exposures while accounting for risk factors that potentially lie on a causal pathway between exposure to abacavir and CVD. RESULTS 11,856 patients were followed for a median of 6.6 years; 365 patients had a CVD event (4.6 events per 1000 patient years). In a conventional Cox model, recent - but not cumulative - exposure to abacavir increased the risk of a CVD event. In the new marginal structural Cox model, continued exposure to abacavir during the past four years increased the risk of a CVD event (hazard ratio 2.06, 95% confidence interval 1.43-2.98). The estimated function for the effect of past exposures suggests that exposure during the past 6 to 36 months caused the greatest increase in risk. CONCLUSIONS Abacavir increases the risk of a CVD event: the effect of exposure is not immediate, rather the risk increases as exposure cumulates over the past few years. This gradual increase in risk is not consistent with a rapidly acting mechanism, such as acute inflammation.

Clinical Practice Recommendations on Genetic Testing of CYP2C9 and VKORC1 Variants in Warfarin Therapy

OBJECTIVE To systematically review evidence on genetic variants influencing outcomes during warfarin therapy and provide practice recommendations addressing the key questions: (1) Should genetic testing be performed in patients with an indication for warfarin therapy to improve achievement of stable anticoagulation and reduce adverse effects? (2) Are there subgroups of patients who may benefit more from genetic testing compared with others? (3) How should patients with an indication for warfarin therapy be managed based on their genetic test results? METHODS A systematic literature search was performed for VKORC1 and CYP2C9 and their association with warfarin therapy. Evidence was critically appraised, and clinical practice recommendations were developed based on expert group consensus. RESULTS Testing of VKORC1 (-1639G>A), CYP2C9*2, and CYP2C9*3 should be considered for all patients, including pediatric patients, within the first 2 weeks of therapy or after a bleeding event. Testing for CYP2C9*5, *6, *8, or *11 and CYP4F2 (V433M) is currently not recommended. Testing should also be considered for all patients who are at increased risk of bleeding complications, who consistently show out-of-range international normalized ratios, or suffer adverse events while receiving warfarin. Genotyping results should be interpreted using a pharmacogenetic dosing algorithm to estimate the required dose. SIGNIFICANCE This review provides the latest update on genetic markers for warfarin therapy, clinical practice recommendations as a basis for informed decision making regarding the use of genotype-guided dosing in patients with an indication for warfarin therapy, and identifies knowledge gaps to guide future research.

Stable coronary artery disease: revascularisation and invasive strategies

Stable coronary artery disease is the most common clinical manifestation of ischaemic heart disease and a leading cause of mortality worldwide. Myocardial revascularisation is a mainstay in the treatment of symptomatic patients or those with ischaemia-producing coronary lesions, and reduces ischaemia to a greater extent than medical treatment. Documentation of ischaemia and plaque burden is fundamental in the risk stratification of patients with stable coronary artery disease, and several invasive and non-invasive techniques are available (eg, fractional flow reserve or intravascular ultrasound) or being validated (eg, instantaneous wave-free ratio and optical coherence tomography). The use of new-generation drug-eluting stents and arterial conduits greatly improve clinical outcome in patients undergoing percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG). PCI is feasible, safe, and effective in many patients with stable coronary artery disease who remain symptomatic despite medical treatment. In patients with multivessel and left main coronary artery disease, the decision between PCI or CABG is guided by the local Heart Team (team of different cardiovascular specialists, including non-invasive and invasive cardiologists, and cardiac surgeons), who carefully judge the possible benefits and risks inherent to PCI and CABG. In specific subsets, such as patients with diabetes and advanced, multivessel coronary artery disease, CABG remains the standard of care in view of improved protection against recurrent ischaemic adverse events.

Republished: Antiplatelet therapy for secondary prevention of coronary artery disease

The choice and duration of antiplatelet therapy for secondary prevention of coronary artery disease (CAD) is determined by the clinical context and treatment strategy. Oral antiplatelet agents for secondary prevention include the cyclo-oxygenase-1 inhibitor aspirin, and the ADP dependent P2Y12 inhibitors clopidogrel, prasugrel and ticagrelor. Aspirin constitutes the cornerstone in secondary prevention of CAD and is complemented by clopidogrel in patients with stable CAD undergoing percutaneous coronary intervention. Among patients with acute coronary syndrome, prasugrel and ticagrelor improve net clinical outcome by reducing ischaemic adverse events at the expense of an increased risk of bleeding as compared with clopidogrel. Prasugrel appears particularly effective among patients with ST elevation myocardial infarction to reduce the risk of stent thrombosis compared with clopidogrel, and offered a greater net clinical benefit among patients with diabetes compared with patients without diabetes. Ticagrelor is associated with reduced mortality without increasing the rate of coronary artery bypass graft (CABG)-related bleeding as compared with clopidogrel. Dual antiplatelet therapy should be continued for a minimum of 1 year among patients with acute coronary syndrome irrespective of stent type; among patients with stable CAD treated with new generation drug-eluting stents, available data suggest no benefit to prolong antiplatelet treatment beyond 6 months.

Benefits and Risks of Extended Duration Dual Antiplatelet Therapy After PCI in Patients With and Without Acute Myocardial Infarction

BACKGROUND The benefits and risks of prolonged dual antiplatelet therapy may be different for patients with acute myocardial infarction (MI) compared with more stable presentations. OBJECTIVES This study sought to assess the benefits and risks of 30 versus 12 months of dual antiplatelet therapy among patients undergoing coronary stent implantation with and without MI. METHODS The Dual Antiplatelet Therapy Study, a randomized double-blind, placebo-controlled trial, compared 30 versus 12 months of dual antiplatelet therapy after coronary stenting. The effect of continued thienopyridine on ischemic and bleeding events among patients initially presenting with versus without MI was assessed. The coprimary endpoints were definite or probable stent thrombosis and major adverse cardiovascular and cerebrovascular events (MACCE). The primary safety endpoint was GUSTO (Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Arteries) moderate or severe bleeding. RESULTS Of 11,648 randomized patients (9,961 treated with drug-eluting stents, 1,687 with bare-metal stents), 30.7% presented with MI. Between 12 and 30 months, continued thienopyridine reduced stent thrombosis compared with placebo in patients with and without MI at presentation (MI group, 0.5% vs. 1.9%, p < 0.001; no MI group, 0.4% vs. 1.1%, p < 0.001; interaction p = 0.69). The reduction in MACCE for continued thienopyridine was greater for patients with MI (3.9% vs. 6.8%; p < 0.001) compared with those with no MI (4.4% vs. 5.3%; p = 0.08; interaction p = 0.03). In both groups, continued thienopyridine reduced MI (2.2% vs. 5.2%, p < 0.001 for MI; 2.1% vs. 3.5%, p < 0.001 for no MI; interaction p = 0.15) but increased bleeding (1.9% vs. 0.8%, p = 0.005 for MI; 2.6% vs. 1.7%, p = 0.007 for no MI; interaction p = 0.21). CONCLUSIONS Compared with 12 months of therapy, 30 months of dual antiplatelet therapy reduced the risk of stent thrombosis and MI in patients with and without MI, and increased bleeding. (The Dual Antiplatelet Therapy Study [The DAPT Study]; NCT00977938).

Development of an Interleukin-1β Vaccine in Patients with Type 2 Diabetes

Interleukin-1β (IL-1β) is a key cytokine involved in inflammatory illnesses including rare hereditary diseases and common chronic inflammatory conditions as gout, rheumatoid arthritis, and type 2 diabetes mellitus, suggesting reduction of IL-1β activity as new treatment strategy. The objective of our study was to assess safety, antibody response, and preliminary efficacy of a novel vaccine against IL-1β. The vaccine hIL1bQb consisting of full-length, recombinant IL-1β coupled to virus-like particles was tested in a preclinical and clinical, randomized, placebo-controlled, double-blind study in patients with type 2 diabetes. The preclinical simian study showed prompt induction of IL-1β-specific antibodies upon vaccination, while neutralizing antibodies appeared with delay. In the clinical study with 48 type 2 diabetic patients, neutralizing IL-1β-specific antibody responses were detectable after six injections with doses of 900 µg. The development of neutralizing antibodies was associated with higher number of study drug injections, lower baseline body mass index, improvement of glycemia, and C-reactive protein (CRP). The vaccine hIL1bQb was safe and well-tolerated with no differences regarding adverse events between patients receiving hIL1bQb compared to placebo. This is the first description of a vaccine against IL-1β and represents a new treatment option for IL-1β-dependent diseases such as type 2 diabetes mellitus (ClinicalTrials.gov NCT00924105).Molecular Therapy (2016); doi:10.1038/mt.2015.227.

A randomised controlled trial: can acupuncture reduce drug requirement during analgosedation with propofol and alfentanil for colonoscopy? A study protocol.

BACKGROUND The number of colonoscopies tremendously increased in recent years and will further rise in the near future. Because of patients' growing expectation on comfort during medical procedures, it is not surprising that the demand for sedation also expands. Propofol in combination with alfentanil is known to provide excellent analgosedation, however, its use is associated with respiratory and cardiovascular depression. Acupuncture could be a technique to reduce drug requirement while providing the same level of sedation and analgesia. METHODS/DESIGN The study will be performed as a single centre, randomised, placebo controlled trial. 153 patients scheduled for propofol/alfentanil sedation during colonoscopy will be randomly assigned to receive electroacupuncture (P6, ST36, LI4), sham acupuncture, or placebo acupuncture. Following endoscopy patients and gastroenterologists have to fill in questionnaires about their sedation experiences. Additionally, patients have to accomplish the Trieger test before and after the procedure. Patient monitoring includes time adapted HR, SpO2, ECG, NIBP, exCO2, OAA/S, and the Aldrete score. The primary outcome parameter is the dosage of propofol necessary for an adequate level of sedation to tolerate the procedure (OAA/S < 4). Effectiveness of sedation, classified by satisfaction levels measured by questionnaires is the secondary outcome parameter. DISCUSSION Moderate to deep sedation using propofol is increasingly applied during colonoscopies with a high satisfaction level among patients despite well-known hemodynamic and respiratory side effects of this hypnotic agent. Acupuncture is known to attenuate gastrointestinal discomfort and pain. We hypothesize that the combination of conventional sedation techniques with acupuncture may result in equally satisfied patients with a lower risk of respiratory and hemodynamic events during colonoscopies. TRIAL REGISTRATION This trial is registered in the Nederland's Trial Register NTR 4325 . The first patient was randomized on 13 February 2014.

Incremental Value of the CRUSADE, ACUITY, and HAS-BLED Risk Scores for the Prediction of Hemorrhagic Events After Coronary Stent Implantation in Patients Undergoing Long or Short Duration of Dual Antiplatelet Therapy

BACKGROUND Multiple scores have been proposed to stratify bleeding risk, but their value to guide dual antiplatelet therapy duration has never been appraised. We compared the performance of the CRUSADE (Can Rapid Risk Stratification of Unstable Angina Patients Suppress Adverse Outcomes With Early Implementation of the ACC/AHA Guidelines), ACUITY (Acute Catheterization and Urgent Intervention Triage Strategy), and HAS-BLED (Hypertension, Abnormal Renal/Liver Function, Stroke, Bleeding History or Predisposition, Labile INR, Elderly, Drugs/Alcohol Concomitantly) scores in 1946 patients recruited in the Prolonging Dual Antiplatelet Treatment After Grading Stent-Induced Intimal Hyperplasia Study (PRODIGY) and assessed hemorrhagic and ischemic events in the 24- and 6-month dual antiplatelet therapy groups. METHODS AND RESULTS Bleeding score performance was assessed with a Cox regression model and C statistics. Discriminative and reclassification power was assessed with net reclassification improvement and integrated discrimination improvement. The C statistic was similar between the CRUSADE score (area under the curve 0.71) and ACUITY (area under the curve 0.68), and higher than HAS-BLED (area under the curve 0.63). CRUSADE, but not ACUITY, improved reclassification (net reclassification index 0.39, P=0.005) and discrimination (integrated discrimination improvement index 0.0083, P=0.021) of major bleeding compared with HAS-BLED. Major bleeding and transfusions were higher in the 24- versus 6-month dual antiplatelet therapy groups in patients with a CRUSADE score >40 (hazard ratio for bleeding 2.69, P=0.035; hazard ratio for transfusions 4.65, P=0.009) but not in those with CRUSADE score ≤40 (hazard ratio for bleeding 1.50, P=0.25; hazard ratio for transfusions 1.37, P=0.44), with positive interaction (Pint=0.05 and Pint=0.01, respectively). The number of patients with high CRUSADE scores needed to treat for harm for major bleeding and transfusion were 17 and 15, respectively, with 24-month rather than 6-month dual antiplatelet therapy; corresponding figures in the overall population were 67 and 71, respectively. CONCLUSIONS Our analysis suggests that the CRUSADE score predicts major bleeding similarly to ACUITY and better than HAS BLED in an all-comer population with percutaneous coronary intervention and potentially identifies patients at higher risk of hemorrhagic complications when treated with a long-term dual antiplatelet therapy regimen. CLINICAL TRIAL REGISTRATION URL: http://clinicaltrials.gov. Unique identifier: NCT00611286.

Mechanisms of Very Late Drug-Eluting Stent Thrombosis Assessed by Optical Coherence Tomography.

BACKGROUND The pathomechanisms underlying very late stent thrombosis (VLST) after implantation of drug-eluting stents (DES) are incompletely understood. Using optical coherence tomography, we investigated potential causes of this adverse event. METHODS AND RESULTS Between August 2010 and December 2014, 64 patients were investigated at the time point of VLST as part of an international optical coherence tomography registry. Optical coherence tomography pullbacks were performed after restoration of flow and analyzed at 0.4 mm. A total of 38 early- and 20 newer-generation drug-eluting stents were suitable for analysis. VLST occurred at a median of 4.7 years (interquartile range, 3.1-7.5 years). An underlying putative cause by optical coherence tomography was identified in 98% of cases. The most frequent findings were strut malapposition (34.5%), neoatherosclerosis (27.6%), uncovered struts (12.1%), and stent underexpansion (6.9%). Uncovered and malapposed struts were more frequent in thrombosed compared with nonthrombosed regions (ratio of percentages, 8.26; 95% confidence interval, 6.82-10.04; P<0.001 and 13.03; 95% confidence interval, 10.13-16.93; P<0.001, respectively). The maximal length of malapposed or uncovered struts (3.40 mm; 95% confidence interval, 2.55-4.25; versus 1.29 mm; 95% confidence interval, 0.81-1.77; P<0.001), but not the maximal or average axial malapposition distance, was greater in thrombosed compared with nonthrombosed segments. The associations of both uncovered and malapposed struts with thrombus were consistent among early- and newer-generation drug-eluting stents. CONCLUSIONS The leading associated findings in VLST patients in descending order were malapposition, neoatherosclerosis, uncovered struts, and stent underexpansion without differences between patients treated with early- and new-generation drug-eluting stents. The longitudinal extension of malapposed and uncovered stent was the most important correlate of thrombus formation in VLST.

Caplacizumab for Acquired Thrombotic Thrombocytopenic Purpura.

BACKGROUND Acquired thrombotic thrombocytopenic purpura (TTP) is caused by aggregation of platelets on ultralarge von Willebrand factor multimers. This microvascular thrombosis causes multiorgan ischemia with potentially life-threatening complications. Daily plasma exchange and immunosuppressive therapies induce remission, but mortality and morbidity due to microthrombosis remain high. METHODS Caplacizumab, an anti-von Willebrand factor humanized single-variable-domain immunoglobulin (Nanobody), inhibits the interaction between ultralarge von Willebrand factor multimers and platelets. In this phase 2, controlled study, we randomly assigned patients with acquired TTP to subcutaneous caplacizumab (10 mg daily) or placebo during plasma exchange and for 30 days afterward. The primary end point was the time to a response, defined as confirmed normalization of the platelet count. Major secondary end points included exacerbations and relapses. RESULTS Seventy-five patients underwent randomization (36 were assigned to receive caplacizumab, and 39 to receive placebo). The time to a response was significantly reduced with caplacizumab as compared with placebo (39% reduction in median time, P=0.005). Three patients in the caplacizumab group had an exacerbation, as compared with 11 patients in the placebo group. Eight patients in the caplacizumab group had a relapse in the first month after stopping the study drug, of whom 7 had ADAMTS13 activity that remained below 10%, suggesting unresolved autoimmune activity. Bleeding-related adverse events, most of which were mild to moderate in severity, were more common with caplacizumab than with placebo (54% of patients vs. 38%). The frequencies of other adverse events were similar in the two groups. Two patients in the placebo group died, as compared with none in the caplacizumab group. CONCLUSIONS Caplacizumab induced a faster resolution of the acute TTP episode than did placebo. The platelet-protective effect of caplacizumab was maintained during the treatment period. Caplacizumab was associated with an increased tendency toward bleeding, as compared with placebo. (Funded by Ablynx; ClinicalTrials.gov number, NCT01151423.).

Increasing Scientific Confidence in Adverse Outcome Pathways: Application of Tailored Bradford-Hill Considerations for Evaluating Weight of Evidence.

Systematic consideration of scientific support is a critical element in developing and, ultimately, using adverse outcome pathways (AOPs) for various regulatory applications. Though weight of evidence (WoE) analysis has been proposed as a basis for assessment of the maturity and level of confidence in an AOP, methodologies and tools are still being formalized. The Organization for Economic Co-operation and Development (OECD) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD 2014a; hereafter referred to as the OECD AOP Handbook) provides tailored Bradford-Hill (BH) considerations for systematic assessment of confidence in a given AOP. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP.

Drug Hypersensitivity: How Drugs Stimulate T Cells via Pharmacological Interaction with Immune Receptors.

Small chemicals like drugs tend to bind to proteins via noncovalent bonds, e.g. hydrogen bonds, salt bridges or electrostatic interactions. Some chemicals interact with other molecules than the actual target ligand, representing so-called 'off-target' activities of drugs. Such interactions are a main cause of adverse side effects to drugs and are normally classified as predictable type A reactions. Detailed analysis of drug-induced immune reactions revealed that off-target activities also affect immune receptors, such as highly polymorphic human leukocyte antigens (HLA) or T cell receptors (TCR). Such drug interactions with immune receptors may lead to T cell stimulation, resulting in clinical symptoms of delayed-type hypersensitivity. They are assigned the 'pharmacological interaction with immune receptors' (p-i) concept. Analysis of p-i has revealed that drugs bind preferentially or exclusively to distinct HLA molecules (p-i HLA) or to distinct TCR (p-i TCR). P-i reactions differ from 'conventional' off-target drug reactions as the outcome is not due to the effect on the drug-modified cells themselves, but is the consequence of reactive T cells. Hence, the complex and diverse clinical manifestations of delayed-type hypersensitivity are caused by the functional heterogeneity of T cells. In the abacavir model of p-i HLA, the drug binding to HLA may result in alteration of the presenting peptides. More importantly, the drug binding to HLA generates a drug-modified HLA, which stimulates T cells directly, like an allo-HLA. In the sulfamethoxazole model of p-i TCR, responsive T cells likely require costimulation for full T cell activation. These findings may explain the similarity of delayed-type hypersensitivity reactions to graft-versus-host disease, and how systemic viral infections increase the risk of delayed-type hypersensitivity reactions.

Uric Acid and Cardiovascular Events: A Mendelian Randomization Study

Obesity and diets rich in uric acid-raising components appear to account for the increased prevalence of hyperuricemia in Westernized populations. Prevalence rates of hypertension, diabetes mellitus, CKD, and cardiovascular disease are also increasing. We used Mendelian randomization to examine whether uric acid is an independent and causal cardiovascular risk factor. Serum uric acid was measured in 3315 patients of the Ludwigshafen Risk and Cardiovascular Health Study. We calculated a weighted genetic risk score (GRS) for uric acid concentration based on eight uric acid-regulating single nucleotide polymorphisms. Causal odds ratios and causal hazard ratios (HRs) were calculated using a two-stage regression estimate with the GRS as the instrumental variable to examine associations with cardiometabolic phenotypes (cross-sectional) and mortality (prospectively) by logistic regression and Cox regression, respectively. Our GRS was not consistently associated with any biochemical marker except for uric acid, arguing against pleiotropy. Uric acid was associated with a range of prevalent diseases, including coronary artery disease. Uric acid and the GRS were both associated with cardiovascular death and sudden cardiac death. In a multivariate model adjusted for factors including medication, causal HRs corresponding to each 1-mg/dl increase in genetically predicted uric acid concentration were significant for cardiovascular death (HR, 1.77; 95% confidence interval, 1.12 to 2.81) and sudden cardiac death (HR, 2.41; 95% confidence interval, 1.16 to 5.00). These results suggest that high uric acid is causally related to adverse cardiovascular outcomes, especially sudden cardiac death.

Oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine that potentially interacts with parasite ferritin and cystatin.

This study investigated the effects of oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine (MEF) and identified proteins that bind to MEF in parasite extracts and human cells by affinity chromatography. In a pilot experiment, MEF treatment was applied 5 days per week and was intensified by increasing the dosage stepwise from 12.5 mg/kg to 200 mg/kg during 4 weeks followed by treatments of 100 mg/kg during the last 7 weeks. This resulted in a highly significant reduction of parasite weight in MEF-treated mice compared with mock-treated mice, but the reduction was significantly less efficacious compared with the standard treatment regimen of albendazole (ABZ). In a second experiment, MEF was applied orally in three different treatment groups at dosages of 25, 50 or 100 mg/kg, but only twice a week, for a period of 12 weeks. Treatment at 100 mg/kg had a profound impact on the parasite, similar to ABZ treatment at 200 mg/kg/day (5 days/week for 12 weeks). No adverse side effects were noted. To identify proteins in E. multilocularis metacestodes that physically interact with MEF, affinity chromatography of metacestode extracts was performed on MEF coupled to epoxy-activated Sepharose(®), followed by SDS-PAGE and in-gel digestion LC-MS/MS. This resulted in the identification of E. multilocularis ferritin and cystatin as MEF-binding proteins. In contrast, when human cells were exposed to MEF affinity chromatography, nicotinamide phosphoribosyltransferase was identified as a MEF-binding protein. This indicates that MEF could potentially interact with different proteins in parasites and human cells.

Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.