Soy Phosphatidylinositol–Containing Lipid Nanoparticle Prolongs the Plasma Survival and Hemostatic Efficacy of B-domain–Deleted Recombinant Canine Factor VIII in Hemophilia A Dogs

Soy phosphatidylinositol (PI) containing lipid nanoparticles prolong plasma survival, improve hemostatic efficacy, and decrease immunogenicity of human B-domain deleted Factor VIII (BDD FVIII) in Hemophilia A (HA) mice. We hypothesize that PI associated BDD FVIII is more potent than the free protein, and using mathematical modeling, have projected that PI associated BDD FVIII could be used for once-weekly prophylactic dosing in patients. To facilitate translation to the clinic, comparative plasma survival and ex vivo efficacy of PI associated recombinant canine FVIII (PI-rcFVIII) were evaluated in HA dogs. 2 HA dogs were administered a 50 U/kg iv dose of free or PI-rcFVIII. rcFVIII activity measurements and ex vivo efficacy analyses like whole blood clotting time (WBCT) and thromboelastography (TEG) were conducted on recovered plasma and whole blood samples. PI association decreased clearance (~25%) and increased plasma exposure (~1.4 fold) of rcFVIII. PI-rcFVIII treated animals had prolonged improvements in WBCTs and TEG parameters compared to free rcFVIII treated animals. Since rcFVIII is a BDD form of FVIII, these studies provide proof-of-principle that observations with human BDD FVIII in mice translate to higher animal species. Additionally, PI-rcFVIII has potential applications in canine HA management and as a bypass therapy in inhibitor-positive HA patients

Porcine and Canine von Willebrand Factor and von Willebrand Disease: Hemostasis, Thrombosis, and Atherosclerosis Studies

Use of animal models of inherited and induced von Willebrand factor (VWF) deficiency continues to advance the knowledge of VWF-related diseases: von Willebrand disease (VWD), thrombotic thrombocytopenic purpura (TTP), and coronary artery thrombosis. First, in humans, pigs, and dogs, VWF is essential for normal hemostasis; without VWF bleeding events are severe and can be fatal. Second, the ADAMTS13 cleavage site is preserved in all three species suggesting all use this mechanism for normal VWF multimer processing and that all are susceptible to TTP when ADAMTS13 function is reduced. Third, while the role of VWF in atherogenesis is debated, arterial thrombosis complicating atherosclerosis appears to be VWF-dependent. The differences in the VWF gene and protein between humans, pigs, and dogs are relatively few but important to consider in the design of VWF-focused experiments. These homologies and differences are reviewed in detail and their implications for research projects are discussed. The current status of porcine and canine VWD are also reviewed as well as their potential role in future studies of VWF-related disorders of hemostasis and thrombosis

ARFI Ultrasound Monitoring of Hemorrhage and Hemostasis In Vivo in Canine Von Willebrand Disease and Hemophilia

A validated method for assessing hemostasis in vivo is critical for testing the hemostatic efficacy of therapeutic agents designed for patients with bleeding disorders such as von Willebrand disease (VWD) and hemophilia A. We hypothesize that rate of bleeding and time to hemostasis can be monitored in vivo by acoustic radiation force impulse (ARFI) ultrasound. We performed ARFI imaging following 12-gauge needle puncture of hind limb muscle encompassing an ~2mm vein in six normal, eight naïve hemophilia A before and after infusing canine factor VIII, three hemophilia A expressing canine factor VIIa following gene transfer, and two naïve VWD dogs. Serial data sets were processed with custom software to (1) estimate the rate of hemorrhage and (2) estimate the time of hemostasis onset. The rate of hemorrhage during the first 30 min following puncture was markedly increased in the VWD dogs relative to normal but was not significantly different between normal, naïve hemophilia A or hemophilia A expressing cFVIIa. ARFI-derived times to hemostasis were significantly longer in naïve hemophilia A dogs than in normal dogs and were shortened by canine coagulation factors VIII and VIIa. These data support our hypothesis that rate of hemorrhage and time to hemostasis in vivo in response to a standardized hemostatic challenge can be detected by ARFI ultrasound in canine models of VWD and hemophilia. These data also suggest that the ARFI-monitored hemostatic challenge is relevant for in vivo testing of the hemostatic efficacy of therapeutic clotting factor replacement products used to treat inherited bleeding disorders

Oxidized LDL and Fructosamine Associated with Severity of Coronary Artery Atherosclerosis in Insulin Resistant Pigs Fed a High Fat/High NaCl Diet

BackgroundInsulin-resistant subjects develop more severe and diffuse coronary artery atherosclerosis than insulin-sensitive controls but the mechanisms that mediate this atherosclerosis phenotype are unknown.Research ObjectiveTo determine the metabolic parameters that associate with the severity of coronary atherosclerosis in insulin resistant pigs fed a high fat/high NaCl diet.Key MethodsThe primary endpoint was severity of coronary atherosclerosis in adult pigs (Sus scrofa, n = 37) fed a high fat diet that also contained high NaCl (56% above recommended levels) for 1 year.Principal FindingsTwenty pigs developed severe and diffuse distal coronary artery atherosclerosis (i.e., severe = intimal area as a percent medial area > 200% in at least 2 coronary artery cross sections and diffuse distal = intimal area as a percent medial area ≥ 150% over 3 sections separated by 2 cm in the distal half of the coronary artery). The other 17 pigs had substantially less coronary artery atherosclerosis. All 37 pigs had blood pressure in a range that would be considered hypertensive in humans and developed elevations in total and LDL and HDL cholesterol, weight gain, increased backfat, and increased insulin resistance (Bergman Si) without overt diabetes. Insulin resistance was not associated with atherosclerosis severity. Five additional pigs fed regular pig chow also developed increased insulin resistance but essentially no change in the other variables and little to no detectible coronary atherosclerosis. Most importantly, the 20 high fat/high NaCl diet -fed pigs with severe and diffuse distal coronary artery atherosclerosis had substantially greater increases (p< 0.05) in oxidized LDL (oxLDL) and fructosamine consistent with increased protein glycation.ConclusionIn pigs fed a high fat/high NaCl diet, glycated proteins are induced in the absence of overt diabetes and this degree of increase is associated with the development of severe and diffuse distal coronary artery atherosclerosis

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A. Haemophilia is a genetic bleeding disorder associated with a deficiency in the coagulation factor VIII. Here, the authors use gene therapy to achieve stable overexpression of factor VIII in platelets of dogs with haemophilia A, preventing the occurrence of severe bleeding episodes for over 2.5 years

Preclinical evaluation of a next-generation, subcutaneously administered, coagulation factor IX variant, dalcinonacog alfa.

IntroductionThe rapid clearance of factor IX necessitates frequent intravenous administrations to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous administration has historically been limited by low bioavailability and potency. Dalcinonacog alfa was developed using a rational design approach to be a subcutaneously administered, next-generation coagulation prophylactic factor IX therapy.AimThis study aimed to investigate the pharmacokinetic, pharmacodynamic, and safety profile of dalcinonacog alfa administered subcutaneously in hemophilia B dogs.MethodsTwo hemophilia B dogs received single-dose daily subcutaneous dalcinonacog alfa injections for six days. Factor IX antigen and activity, whole blood clotting time, and activated partial thromboplastin time were measured at various time points. Additionally, safety assessments for clinical adverse events and evaluations of laboratory test results were conducted.ResultsThere was an increase in plasma factor IX antigen with daily subcutaneous dalcinonacog alfa. Bioavailability of subcutaneous dalcinonacog alfa was 10.3% in hemophilia B dogs. Daily subcutaneous dosing of dalcinonacog alfa demonstrated the effects of bioavailability, time to maximal concentration, and half-life by reaching a steady-state activity sufficient to correct severe hemophilia to normal, after four days.ConclusionThe increased potency of dalcinonacog alfa facilitated the initiation and completion of the Phase 1/2 subcutaneous dosing study in individuals with hemophilia B

Preclinical evaluation of a next-generation, subcutaneously administered, coagulation factor IX variant, dalcinonacog alfa.

IntroductionThe rapid clearance of factor IX necessitates frequent intravenous administrations to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous administration has historically been limited by low bioavailability and potency. Dalcinonacog alfa was developed using a rational design approach to be a subcutaneously administered, next-generation coagulation prophylactic factor IX therapy.AimThis study aimed to investigate the pharmacokinetic, pharmacodynamic, and safety profile of dalcinonacog alfa administered subcutaneously in hemophilia B dogs.MethodsTwo hemophilia B dogs received single-dose daily subcutaneous dalcinonacog alfa injections for six days. Factor IX antigen and activity, whole blood clotting time, and activated partial thromboplastin time were measured at various time points. Additionally, safety assessments for clinical adverse events and evaluations of laboratory test results were conducted.ResultsThere was an increase in plasma factor IX antigen with daily subcutaneous dalcinonacog alfa. Bioavailability of subcutaneous dalcinonacog alfa was 10.3% in hemophilia B dogs. Daily subcutaneous dosing of dalcinonacog alfa demonstrated the effects of bioavailability, time to maximal concentration, and half-life by reaching a steady-state activity sufficient to correct severe hemophilia to normal, after four days.ConclusionThe increased potency of dalcinonacog alfa facilitated the initiation and completion of the Phase 1/2 subcutaneous dosing study in individuals with hemophilia B

Fasting oxLDL Levels (units/L).

<p>*Wilcoxon signed rank statistic for change from baseline within group.</p><p>^†Wilcoxon rank sum statistic for differences between groups.</p><p>Fasting oxLDL Levels (units/L).</p

Coronary Artery Atherosclerosis in Insulin Resistant Pigs.

<p>Panels A1 to A5: Representative serial sections from the proximal to distal left anterior descending coronary artery are shown from an IR pig with severe and diffuse distal coronary atherosclerosis. The mean intimal area as a percent of medial area for all three coronary arteries for this pig was 203.6% proximally and 338.5% distally. Panels B1 to B5: In contrast, a pig with moderate coronary atherosclerosis had a mean intimal area as a percent of medial area for all three coronary arteries of 11.3% proximally and 5.8% distally. Features of coronary atherosclerosis in rows A and B are shown at higher magnification in rows C and D, respectively: intimal thickening (IT) and calcification in a pig with moderate atherosclerosis (Ca²⁺ (moderate), Panels B1 and D1), calcification in a pig with severe atherosclerosis (Ca²⁺ (severe), Panels A4 and C2), fibrous cap (FC, Panel A2 and C1), hemorrhage into the plaque (H, Panels A4 and C3), medial thinning (MT, Panels A4 and D2), necrosis (N, Panels A4 and D3). (Hematoxylin and Eosin, mag bar = 500 μm is shown in B5 for rows A and B; mag bar = 10 μm in all panels in rows C and D).</p

Intimal Area as % Medial Area from Serial Coronary Artery Sections.

<p>The mean ± SD for intimal area as % medial area of all sections from all three coronary arteries are shown by severe and diffuse (black), moderate (white), or control (grey) groups. Sections were taken at 1 cm intervals. The solid black vertical line indicates the division between the proximal and distal halves of each coronary artery. The horizontal black line at 200% indicates the criteria for severe coronary atherosclerosis. The hashed line at 150% indicates diffuse and distal coronary atherosclerosis when it occurs over 3 sections (i.e., 2 cm). Using these criteria, pigs in the severe atherosclerosis group also exhibited diffuse and distal coronary atherosclerosis in all three coronary arteries.</p