A lipase fusion feasts on fat

The enzyme lipoprotein lipase (LPL) is responsible for breaking down triglycerides in the blood. Mutations in LPL cause a rare but debilitating disorder characterized by excessive plasma triglyceride levels for which treatment options are limited. Nimonkar et al. now present a fusion protein between LPL and its physiological transporter GBIHBP1 that is highly active and largely resistant to physiological inhibitors of LPL. Injecting this fusion protein effectively lowers plasma triglycerides in mice and represents a promising new approach for lowering triglycerides in patients with familial chylomicronemia syndrome.

Refrigerated Platelets Are Superior Compared to Standard-of-Care and Respond to Physiologic Control Mechanisms Under Microfluidic Flow Conditions

Background: Platelets are required for effective treatment of severe hemorrhage. Standard-of-care storage is at room temperature (RT), but leads to a storage lesion characterized by loss of hemostatic function and increased risk of bacterial contamination. Refrigeration (4C) mitigates adverse effects; however, it results in pre-activation or priming, which may be suggestive of prothrombotic tendencies. We previously showed that 4C-stored platelets (4C-PLT) retain responses to physiological inhibitors comparable to those of fresh platelets (FR-PLT) in two static aggregation assays. To more closely mimic in-vivo conditions, we tested adhesive response in a microfluidic environment under physiologic high-shear flow. We hypothesized that 4C-PLT display superior adhesion compared to standard-of-care (RT-PLT) and that platelet hemostatic inhibition due to prostacyclin and nitric oxide (NO) would be similar to fresh. Methods: Apheresis platelets (AP) collected from 4 healthy donors were stored for 5 days at RT (22-24°C) or 4C (1-6°C). Additional whole blood was collected to obtain red blood cells (RBCs). Platelet samples were assayed on Day 1 (fresh) and Day 5 (RT-PLT and 4C-PLT) in the presence or absence of prostacyclin (10 nM Prostaglandin I2, PGI2) or an NO donor (50 uM S-Nitrosoglutathione, GSNO). Bioflux plates (Fluxion) were coated with 100 µg/ml type-1 collagen. Prior to perfusion, platelets were stained with calcein-AM (300x103PLT/ul) and RBCs were added to a hematocrit of 40%. Samples were perfused through the collagen-coated wells at an arterial shear rate of 720s-1, and compared to bovine serum album (BSA)-coated channels as a control to assess nonspecific binding. A fluorescence microscope acquired images every 30 sec for 6 min. Data were reported as fluorescence intensity units (FIU) and surface coverage (SC%) measured with Bioflux Montage (MetaMorph) software. Data were analyzed using two-way ANOVA and a post hoc Tukey test for multiple comparisons. Significance was p<0.05. Results: The perfusion images illustrate that 4C-PLT adhesion was similar to FR-PLT and demonstrably better than RT-PLT (Fig.1A). Measured fluorescence confirmed visual findings (Fig. 1B-E) and demonstrated that RT-PLT adhesion was considerably attenuated. Cold-stored platelet average SC% after 6 min of perfusion was similar to fresh (4C-PLT: 4.5±0.8%; FR-PLT: 4.7±0.8%; p=0.86; Figure 1F), but that of RT-PLT was significantly decreased (RT-PLT: 1.2±0.2%; p<0.001; Figure 1F). The addition of PGI2 to all groups reduced adhesion to collagen-coated surfaces under high shear (FR-PLT: 1.6±0.3%; p<0.001; 4C-PLT: 1.5±0.5%; p<0.01; RT-PLT: 0.5±0.1%; p≥0.05). GSNO treatment was similar, although reductions were not statistically significant (FR-PLT: 3.2±0.6%; 4C-PLT: 3.2±0.8%; RT-PLT: 0.8±0.2%; p≥0.05). Conclusion: Although 4C-stored platelets show increased levels of activation compared to fresh, they retain comparable responses to physiological inhibitors under shear flow. Furthermore, standard-of-care RT-stored platelets are unable to adhere to collagen under flow conditions. These data suggest that 4C platelets, although primed and hemostatically more active, may respond to homeostatic signals in vivo and may not pose a risk of promoting unregulated clot formation. RT-PLT function is significantly reduced and did not recover activity in this physiologically relevant model. Testing 4C-PLT in a small animal model of trauma could further elucidate these findings. Figure 1. Platelet adhesion under high shear with and without inhibitors. (A) Micrographs of FR-PLT, 4C-PLT, and RT-PLT samples are shown for a single donor after 6 minutes of perfusion. Representative fluorescence intensity unit (FIU) traces are shown for (B) FR-PLT, (C) 4C-PLT, and (D) RT-PLT (Untreated •, PGI2 ■, and GSNO ▲). Data for (E) FIU (n=4) and (F) SC% (n=4) after 360 s are mean ± SEM (Untreated = black, PGI2= gray, and GSNO = white). The micrographs were cropped and enlarged; however image size proportionality was maintained between groups and timepoints. * p<0.05, † p<0.01, and ‡ p<0.001, and § p<0.0001 Figure 1. Platelet adhesion under high shear with and without inhibitors. (A) Micrographs of FR-PLT, 4C-PLT, and RT-PLT samples are shown for a single donor after 6 minutes of perfusion. Representative fluorescence intensity unit (FIU) traces are shown for (B) FR-PLT, (C) 4C-PLT, and (D) RT-PLT (Untreated •, PGI2 ■, and GSNO ▲). Data for (E) FIU (n=4) and (F) SC% (n=4) after 360 s are mean ± SEM (Untreated = black, PGI2= gray, and GSNO = white). The micrographs were cropped and enlarged; however image size proportionality was maintained between groups and timepoints. * p<0.05, † p<0.01, and ‡ p<0.001, and § p<0.0001 Disclosures No relevant conflicts of interest to declare.

Mod-5014, a Novel Long-Acting FVIIa Proposing An Improved Prophylactic and On Demand Treatment For Hemophilic Patients Following SC and IV Administration – Comprehensive In -Vitro and In -Vivo evaluation In Preparation For Clinical Studies

Introduction Prolor Biotech Inc. is a clinical stage public company developing biobetter long acting versions of existing therapeutic proteins utilizing a technology termed CTP. The technology involves fusion of the C terminus peptide of hCG to one or both ends of the target protein. The technology was clinically validated and proven as a safe and efficient way for prolonging the half-lives of several therapeutic proteins while maintaining their biological activity. The aim of this extensive study was to characterize the in vitro potency as well as the in –vitro interactions of MOD-5014 (FVIIa-CTP ) with physiological inhibitors and cofactors and to determine its pharmacokinetic (PK), pharmacodynamic (PD) and long term hemostatic effects in the relevant hemophilic animal models following IV and SC administrations as moving forward into clinical studies . Methods FVII-CTP was expressed in CHO cells, purified and activated utilizing a CTP specific purification process. MOD-5014 interactions with physiological inhibitors and cofactors was characterized in –vitro utilizing SPR and ex-vivo assays ( TEG and TG). In order to assess the long term in vivo effect , MOD-5014 was administered to warfarin treated rats or FVIII-/- mice, and following IV and SC injection the PK and PD profiles were determined as well as coagulation parameters (PT,aPTT ,TG and FVIIa activity) at a time dependent manner. In addition, the long term hemostatic effect was evaluated following bleeding challenge by tail clip assay and tail vein transection as compared to commercial rFVIIa. Results MOD-5014 in vitro activity was comparable to commercial FVIIa. PT , thrombin generation , PK and PD parameters following IV and SC administration were superior to those of rFVIIa. PT values of warfarin treated rats were maintained normal for significantly longer time post injection. MOD-5014 half-life and AUC following IV administration were 5 and 3.5 fold higher, respectively and were also significantly superior following SC injection. Following SC administration, MOD-5014's bioavailability, was shown to be superior to commercial rFVIIa in both rats and hemophilic mice models as well. In a tail vein transection studies, MOD-5014 had a profound effect on survival rate, which was maintained for more than 24 and significantly reduced duration and intensity of bleeding was also observed in tail clip studies in warfarin treated rats as well as in hemophilic mice in both routes. Finally, toxicological studies in rodents demonstrated that MOD-5014 is safe and tolerable at relatively high doses. Conclusion Attachments of CTP to FVIIa led to a markedly enhanced PK, increased exposure as reflected by AUC, improved recovery and a prolonged hemostatic effect in hemophilic mice and rats further supporting the comparable specific activity of MOD-5014 to rFVIIa. In addition, SC administration of MOD-5014 resulted in improved bioavailability and exposure was significantly prolonged relative to IV administration which was also translated to superior-vivo efficacy. CTP attachment to FVIIa had no significant impact on the in-vitro interactions with physiological inhibitors and cofactors proposing a similar mechanism of action and comparable activity. Our data suggest that CTP fused FVIIa is safe and tolerable in rodents and has the potential to significantly the frequency of injection given on demand as well as potentially enable prophylactic treatment for hemophilic patients. Disclosures: No relevant conflicts of interest to declare.