Paroxetine substantially decreases intraplatelet serotonin content and thereby reduces platelet plug formation under shear stress, and responsiveness to thrombin receptor activating peptide-induced platelet activation. Further studies will reveal whether these pharmacodynamic effects can be exploited for treatment of thrombotic artery disease.

Bleeding due to impaired platelet function is a major side effect of the Bruton´s tyrosine kinase (BTK) inhibitor ibrutinib. We quantitatively assessed ristocetin-induced platelet aggregation (RIPA) in 64 patients with chronic lymphocytic leukemia (CLL) under ibrutinib at 287 time points. Eighty-seven bleeding episodes in 39 patients were registered (85 CTC grade 1 or 2, 2 CTC grade 3) during a median observation period of 10.9 months. At times of bleeding, RIPA values were significantly lower (14 vs. 28 U; p<0.0001). RIPA was impaired in patients receiving concomitant anti-platelet therapy or anticoagulation (14 U vs. 25 U, p=0.005). A gradual decline of median RIPA values was observed with increasing bleeding severity. Importantly, no CTC grade 2 or 3 bleeding were observed with RIPA values above 36 U. Sequential monitoring indicated a decrease of RIPA values from a median of 17 to 9 U within 2 weeks after initiation of treatment as well as an increase above the critical threshold of 36 U within 7 days when ibrutinib was paused. Low RIPA values were similar during treatment with another BTK inhibitor, CC292. Quantitative assessment of platelet function is a practical tool to monitor bleeding tendency under BTK-inhibitor therapy.

MCP-1 levels are about 30 percent higher in men than in premenopausal women, and MCP-1 levels are also higher in persons with RBCs expressing Fy antigens than in Fy(a-b-) persons. These findings have direct implications for the concept and interpretation of clinical studies measuring MCP-1 levels; the role of the observed differences in MCP-1 levels for the pathogenesis of MCP-1-dependent diseases, such as atherosclerosis, merits further investigation.

The previously described anti-endotoxin effect of colistin has not been investigated in humans yet. We performed a randomized, double-blind, placebo-controlled crossover trial to determine the degree of colistin-driven modulation of inflammatory response in blood of lipopolysaccharide (LPS)-challenged healthy volunteers in a human endotoxemia model. After a single intravenous dose of 2.5 million IU colistin methanesulfonate, interleukin (IL)-6, IL-8, tumor necrosis factor alpha (TNF-α), and IL-1β concentrations as well as other biomarkers of inflammation such as C-reactive protein, differential leukocyte counts, and body temperature were measured up to 24 h postdose. Colistin significantly decreased the inflammatory cytokine response to LPS in blood of healthy volunteers. This effect was most evident for IL-6, IL-8, and TNF-α. This study is the first to confirm the anti-endotoxin effect of colistin in humans in vivo. Further studies might increase our knowledge on the interaction between colistin and the effectors of the immune system.

A protective role against atherosclerosis can be attributed to angiotensin converting enzyme inhibitors (ACE-I), since they have been shown to reduce mortality in patients at cardiovascular risk. Since plasma levels of adhesion molecules are considered surrogate markers of endothelial cell activation and atherogenesis, we compared the levels of adhesion molecules after treatment with the ACE-I enalapril or the direct angiotensin- receptor antagonist losartan or placebo. In a randomized, controlled trial, 21 hypercholesterolemic volunteers received 50 mg/d losartan or 20 mg/d enalapril or placebo for twelve weeks. Plasma levels of circulating intercellular adhesion molecule-1 (cICAM-1), vascular adhesion molecule-1 (cVCAM-1), and E-selectin (cE-SEL) were measured by ELISA. Surface expression of ICAM-1 on circulating leukocytes was determined by flow cytometry. Enalapril and losartan but not placebo induced a small but stable decrease of cICAM-1 and cVCAM-1, while cE-SEL and leukocyte expression of ICAM-1 remained unchanged. The lowering of plasma adhesion molecules may indicate an antiatherogenic effect of angiotensin II blockade in hypercholesterolemia. While such preventive effect will have to be proven in clinical trials, our results do not support a preference for either enalapril or losartan with regard to their possible vasoprotective role.

No abstract

Background. Inhibition of Bruton´s tyrosine kinase (BTK) with the small molecule ibrutinib has significantly improved the survival of patients with chronic lymphocytic leukemia (CLL). BTK is also expressed in platelets. Collagen- and von Willebrand Factor (vWF)-dependent (ristocetin-induced) impairment of platelet function has recently been described (Levade M et al., Blood 2014, 124:3991-5;Kamel S et al., Leukemia 2015, 29:783-787) . Bleeding events were observed in 61% of patients in a recently published 3 year follow-up (Byrd JC et al., Blood 2015, 125:2497-2506). Bleeding under ibrutinib is generally mild (CTC grade 1-2 corresponding to spontaneous bruising or petechiae), but grade 3 or 4 bleeding can be observed, particularly after trauma. We hypothesized that quantitative assessment of platelet aggregation in ibrutinib CLL patients could help (1) to predict bleeding tendency, and (2) to guide patients through invasive procedures. Patientsand Methods. Twenty-four adult patients with previously treated CLL (16 male/8 female, median age 67 years, range 55-84) received ibrutinib orally at a planned dose of 420mg/day and were regularly monitored and thoroughly investigated for bleeding tendency. The median time on ibrutinib was 7.5 months, (range 1-27). Bleeding events (any CTC grade) occurred in 13 (54%) and dose-reductions to 280 (N=12) or 140mg (N=3) (for bleeding, infections, or neutropenia) were made in 15 (63%) of patients during a median observation period of 5 months (range 1-12). Bleeding was observed in 4 of 6 patients with concomitant anticoagulation. Of note, only 1 of the 24 patients had a CTC grade 3 bleeding event, and no grade 4 or 5 events were observed. Ristocetin-induced platelet aggregation (RIPA, herein referred to as RCoF) was quantitatively measured in fresh hirudin-blood by whole blood aggregometry with a Multiplate® Analyzer (Roche Diagnostics). Platelet aggregation was expressed in AUC units (U) (normal range 98-180U). Controls included normal subjects (N=53). Consecutive samples before and during treatment were available in all patients. Statistical methods comprised t-Test and ANOVA using SAS. Results. Ristocetin-induced platelet aggregation was already diminished before ibrutinib treatment (median 51 RCoF U) when compared to normal controls (Table 1). This is likely due to lower platelet counts in CLL patients influencing overall platelet aggregability (Hanke AA et al., Eur J Med Res 2010, 15:214-219). During ibrutinib treatment, platelet aggregation was substantially impaired (median of 22U). A direct comparison of available paired samples in 5 patients showed a significant decrease after ibrutinib initiation (51 to 14.5U; p=0.0028). Of note, significantly lower values were measured at visits when bleeding events were documented (N=34) compared to patient visits without bleeding tendency (N=70) (median 13 vs. 42U; p<0.001). The median RCoF value was lower in patients with CTC grade > 2 (N=10) vs. <2 bleeding (11 vs. 14U). Similar results were obtained for collagen-dependent platelet function (bleeding vs. no bleeding: 17 vs. 19.5U; p=0.002). RCoF values were correlated with platelet count (r2 =0.34; p<0.0001) at median values of 103 vs. 138 G/L in patients with or without bleeding, respectively. There was also a significant difference between the lowest RCoF values in individual patients with or without bleeding (7.5 vs. 16.5U; p=0.027) (Figure 1). No bleeding event was observed in patients whose lowest RCoF value was greater than 25U. Long-term kinetics of vWF-dependent platelet function was assessed in 7 patients and corresponded with ibrutinib dose. When ibrutinib was stopped, recovery of RCoF to greater than 70U was observed in as little as 48hours, suggesting a short time to normalization of platelet function. Conclusion. These data indicate that quantitative assessment of vWF-dependent platelet function in ibrutinib treated patients may serve to monitor therapy particularly in the setting of bleeding tendency, anticoagulation, or planned invasive procedures. Further evaluation of platelet function as a pharmacodynamic marker seems warranted. Figure 1. VWF-dependent platelet function (RCoF) in normal subjects or CLL patients before and during ibrutinib treatment with or without bleeding. Figure 1. VWF-dependent platelet function (RCoF) in normal subjects or CLL patients before and during ibrutinib treatment with or without bleeding. Figure 2. Lowest RCoF values in individual patients with or without bleeding. Figure 2. Lowest RCoF values in individual patients with or without bleeding. Disclosures Staber: Genactis: Research Funding; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Takeda-Millenium: Research Funding; Karyopharm: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria. Pabinger:Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Baxter: Membership on an entity's Board of Directors or advisory committees. Jilma:True North Therapeutics, Inc.: Consultancy, Research Funding. Jaeger:Hoffmann La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; True North Therapeutics, Inc.: Research Funding.

Autoantibody mediated classical complement pathway (CP) activation has been hypothesized to drive hemolytic anemia in cold agglutinin disease (CAD) patients. Red blood cell (RBC) destruction is believed to occur as a result of C3 opsonin mediated extravascular hemolysis in the liver. We recently reported that TNT009, a humanized monoclonal antibody targeting the CP specific serine protease C1s, rapidly restores hemoglobin levels in severely anemic CAD patients. Here we describe the pharmacodynamic changes in the complement profile of patients to provide a mechanistic understanding of the hematological responses and therapeutic benefit observed following TNT009 treatment. In a Phase 1b trial, we enrolled 5 female CAD patients with severe anemia, one of whom had a lymphoplasmacytic lymphoma with >70% bone marrow infiltration and no measurable CP activity prior to dosing. This patient did not respond to TNT009 while on study and will be omitted from subsequent analyses. Patients were given an initial 10 mg/kg test dose of TNT009 on Day 1, followed by four weekly doses of 60 mg/kg on Day 2 or Day 5. Patients were followed for 4 weeks following the last dose (washout). Plasma and serum samples were collected throughout the study to measure TNT009 concentrations and to monitor serological markers of anemia and hemolysis. Additionally, futhan-containing plasma samples were collected to assess the levels of CP specific components including C1s, C1s-C1INH, and C1q by ELISA. RBCs were collected to monitor cell surface complement deposition (C3 fragments) via flow cytometry. Finally, C4 levels and an ELISA-based readout of CP activity were examined as measures of the pharmacodynamic effect of TNT009. Baseline levels of circulating C4 were either low or undetectable in CAD patients. Accordingly, serum CP activity was reduced compared to normal human serum samples. Following the first 60 mg/kg TNT009 dose, CP activity was immediately and completely inhibited within 15 minutes of dosing in all patients and remained inhibited for 3 weeks after the last dose. During this period of inhibition, C4 levels rose from a median circulating concentration of <90 mcg/mL (range: <70 - 145) to 251 mcg/mL (range: 238 - 353; p < .001). Plasma C1s levels, on the other hand, decreased from a median plasma concentration of 53.3 mcg/mL (range: 49.4 - 60.3) to a nadir of <3.13 mcg/mL, the lower limit of quantification (LLOQ), (p < .001). Similarly, C1s-C1INH decreased from a median value of 4.5 mcg/mL (range: 4.2 - 5.3) to a nadir of <0.16 mcg/mL (LLOQ; p < .001). Notably, circulating plasma C1q levels were unaffected. Classical pathway inhibition led to a significant increase in reticulocytes in all patients by, on average, 69% within 24 hours of dosing (p < .05). Interestingly, within 1 week after the first TNT009 dose, reticulocyte counts returned to pre-treatment levels and continued to decrease throughout the study, as expected when hemoglobin normalizes. Similarly, within 24 hours of the first dose of TNT009, bilirubin levels dropped from a median value of 2.1 mg/dL (range 1.6 - 3.8) to 0.7 mg/dL (range 0.6 - 1.2), resulting in an average reduction of 66% from baseline levels (p < .05) and returning to pre-treatment levels following washout. Finally, we monitored in vivo complement activation by staining for C3 fragment deposition on RBCs. In general, we observed a gradual reduction in the percentage of C3 fragment positive RBCs over the course of the study from a median value of 49% (range: 37 - 81) to 29% (range: 20 - 38) before washout of TNT009. The decrease in opsonized RBCs was concomitant with the rise in hemoglobin (example shown in Figure 1). Figure 1: Elevation of hemoglobin is associated with a decrease in C3 fragment coated erythrocytes Here we report that TNT009 administration depletes circulating C1s and immediately halts in vivo CP activity, normalizing plasma C4 levels in CAD patients. The abrupt increase in reticulocyte count within 24 hours of dosing suggests that cold agglutinin mediated complement activation affects reticulocyte survival, preventing their maturation into erythrocytes. The observed reduction in C3 opsonized RBCs suggests that TNT009 ameliorates anemia by preventing complement mediated hepatic RBC sequestration, supported by the immediate normalization of circulating bilirubin levels. These results provide a mechanistic interpretation of the therapeutic effects of TNT009 in CAD patients. Figure 1. Figure 1. Disclosures Panicker: True North Therapeutics, Inc.: Employment, Equity Ownership. Hussain:True North Therapeutics, Inc.: Employment, Equity Ownership. Parry:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Gilbert:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Jaeger:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.