N-acetylcysteine in preclinical mouse and baboon models of thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder diagnosed by thrombocytopenia and hemolytic anemia, associated with a deficiency in von Willebrand factor (VWF)-cleaving protease ADAMTS13. Current treatment is based on plasma infusion for congenital TTP, or plasma exchange, often in combination with immunosuppressive agents, for acquired TTP. These treatment methods are not always effective; therefore, new treatment methods are highly necessary. N-acetylcysteine (NAC), an FDA-approved anti-mucolytic agent, is a possible new treatment strategy for TTP, as it was demonstrated to reduce disulfide bonds in VWF, thereby decreasing VWF multimers size and hence their prothrombotic potential. We investigated whether NAC, without concurrent plasma exchange and immunosuppressive therapy, is effective in preventing and resolving TTP signs, using well-established murine and baboon models for TTP. In mice, prophylactic administration of NAC was effective in preventing severe TTP signs. This in vivo finding was supported by in vitro data demonstrating the VWF multimer-reducing properties of NAC in solution. Nonetheless, in both mice and baboons, administration of NAC was not effective in resolving preexisting TTP signs; thrombocytopenia, hemolytic anemia, and organ damage could not be reversed, as thrombus resolution was not achieved. Failure to improve clinical outcome occurred even though a reduction in VWF multimers was observed, demonstrating that NAC was efficient in reducing disulfide bonds in circulating VWF multimers. In conclusion, prophylactic administration of NAC, without concurrent plasma exchange, was effective in preventing severe TTP signs in mice, but NAC was not effective in resolving preexistent acute TTP signs in mice and baboons.

Dual inhibition of thrombin and activated factor X attenuates disseminated intravascular coagulation and protects organ function in a baboon model of severe Gram-negative sepsis.

BACKGROUND: Inhibition of procoagulant pathways may improve outcome in sepsis. We examined whether a dual short-acting thrombin (factor II) and factor X (FX)a inhibitor (SATI) ameliorates sepsis-induced disseminated intravascular coagulation (DIC) and is organ-protective. METHODS: Escherichia coli were infused for 2 h in 22 anesthetized baboons. The control (CO) group (n = 8) received sterile isotonic solution only. In the treatment groups, SATI was administered starting 15 minutes after the end of the bacterial exposure. In the low-dose group (LD-SATI, n = 8), SATI was infused with 75 µg/kg/h for the first hour, followed by 23 µg/kg/h until the end of the study. In the high-dose SATI group (HD-SATI, n = 6), 225 µg/kg/h was administered for the first hour followed by continuous infusion of 69 µg/kg/h until termination of the study. RESULTS: Sepsis-induced DIC was attenuated, as reflected by lower peak thrombin-antithrombin complexes (threefold) and D-dimer levels (twofold) in both SATI groups compared to the CO. This coincided with strongly improved cell/organ protection assessed by decreased levels of lactate dehydrogenase (threefold), creatinine (twofold), aspartate aminotransferase (threefold), and amylase (twofold) compared to the CO group. Anuria, which started at 8 h in the CO group, was prevented in both SATI groups. Peak interleukin-6 release at 12 h was prevented in the treatment groups. In both SATI groups, fewer catecholamines were necessary and no bleeding complications were observed. CONCLUSIONS: Dual inhibition of thrombin and FXa preserved activation of coagulation, protected organ function and ameliorated inflammation in severe Gram-negative sepsis in baboons. SATI could be a novel therapeutic agent against sepsis-induced DIC.

The Cape Chacma baboon is not suitable for evaluating human targeted anti-GPVI agents.

An effective and safe anti-platelet drug is central to the management of patients with acute coronary syndrome (ACS). Glycoprotein VI (GPVI) is currently regarded as a potential target for novel anti-platelet agents due to its collagen-binding potential. Development of anti-thrombotics is associated with testing in animals. We have previously successfully evaluated anti-platelet drugs in the Cape Chacma baboon (Papio ursinus). However, various anti-GPVI agents did not have an effect on baboons when evaluated in our arterial thrombosis model. To evaluate the suitability of baboons for GPVI studies, we performed collagen-induced platelet aggregation, GPVI quantification and DNA sequencing. Baboon platelets needed double the amount of collagen compared to human platelets to illicit proper aggregation. GPVI quantification was unsuccessful due to non-binding of monoclonal antibodies. Sequencing of the GPVI gene revealed 36 SNPs leading to 14 amino acid changes, as well as a 9 bp deletion causing a 3 amino acid deletion. Several of the amino acid changes were within the ligand binding region of GPVI, causing limited binding of humanized anti-GPVI antibodies to the baboon platelets. Therefore, the baboon was deemed not suitable to evaluate human targeted anti-GPVI agents.

Evaluation of efficacy and safety of the anti-VWF Nanobody ALX-0681 in a preclinical baboon model of acquired thrombotic thrombocytopenic purpura.

ALX-0681 is a therapeutic Nanobody targeting the A1-domain of VWF. It inhibits the interaction between ultra-large VWF and platelet GpIb-IX-V, which plays a crucial role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In the present study, we report the efficacy and safety profile of ALX-0681 in a baboon model of acquired TTP. In this model, acute episodes of TTP are induced by administration of an ADAMTS13-inhibiting mAb. ALX-0681 completely prevented the rapid onset of severe thrombocytopenia and schistocytic hemolytic anemia. After induction of TTP, platelet counts also rapidly recovered on administration of ALX-0681. This effect was corroborated by the full neutralization of VWF activity. The schistocytic hemolytic anemia was also halted and partially reversed by ALX-0681 treatment. Brain CT scans and post mortem analysis did not reveal any sign of bleeding, suggesting that complete neutralization of VWF by ALX-0681 under conditions of thrombocytopenia was not linked with an excessive bleeding risk. The results obtained in this study demonstrate that ALX-0681 can successfully treat and prevent the most important hallmarks of acquired TTP without evidence of a severe bleeding risk. Therefore, ALX-0681 offers an attractive new therapeutic option for acquired TTP in the clinical setting.

Inhibition of von Willebrand factor-platelet glycoprotein Ib interaction prevents and reverses symptoms of acute acquired thrombotic thrombocytopenic purpura in baboons.

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) can be explained by the absence of active ADAMTS13, leading to ultra-large von Willebrand factor (UL-VWF) multimers spontaneously interacting with platelets. Preventing the formation of UL-VWF-platelet aggregates therefore is an attractive new treatment strategy. Here, we demonstrate that simultaneous administration of the inhibitory anti-VWF monoclonal antibody GBR600 and the inhibitory anti-ADAMTS13 antibody 3H9 to baboons (prevention group) precluded TTP onset as severe thrombocytopenia and hemolytic anemia were absent in these animals. In addition, partial VWF inhibition was not enough to prevent thrombocytopenia, demonstrating the specificity of this therapeutic strategy. GBR600 treatment of baboons during acute TTP (treatment group) resulted in a rapid recovery of severe thrombocytopenia similar to the platelet count increases observed in TTP patients treated by plasma exchange. Baboons in the control group only injected with 3H9 developed early stages of TTP as previously described. Hence, inhibiting VWF-GPIb interactions is an effective way to prevent and treat the early symptoms of acquired TTP in baboons.

Antithrombotic drug candidate ALX-0081 shows superior preclinical efficacy and safety compared with currently marketed antiplatelet drugs.

Neutralizing the interaction of the platelet receptor gpIb with VWF is an attractive strategy to treat and prevent thrombotic complications. ALX-0081 is a bivalent Nanobody which specifically targets the gpIb-binding site of VWF and interacts avidly with VWF. Nanobodies are therapeutic proteins derived from naturally occurring heavy-chain-only Abs and combine a small molecular size with a high inherent stability. ALX-0081 exerts potent activity in vitro and in vivo. Perfusion experiments with blood from patients with acute coronary syndrome on standard antithrombotics demonstrated complete inhibition of platelet adhesion after addition of ALX-0081, while in the absence of ALX-0081 residual adhesion was observed. In a baboon efficacy and safety model measuring acute thrombosis and surgical bleeding, ALX-0081 showed a superior therapeutic window compared with marketed antithrombotics. Pharmacokinetic and biodistribution experiments demonstrated target-mediated clearance of ALX-0081, which leads to a self-regulating disposition behavior. In conclusion, these preclinical data demonstrate that ALX-0081 combines a high efficacy with an improved safety profile compared with currently marketed antithrombotics. ALX-0081 has entered clinical development.

Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon (Papio ursinus).

Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factor-cleaving protease (ADAMTS13) has been demonstrated, but additional genetic and/or environmental triggers are thought to be required to incite acute illness. Here we report that 4 days of ADAMTS13 functional inhibition is sufficient to induce TTP in the baboon (Papio ursinus), in the absence of inciting triggers because injections with an inhibitory monoclonal antibody (mAb) consistently (n = 6) induced severe thrombocytopenia (< 12 × 10(9)/L), microangiopathic hemolytic anemia, and a rapid rise in serum lactate dehydrogenase. Immunohistochemical staining revealed the characteristic disseminated platelet- and von Willebrand factor-rich thrombi in kidney, heart, brain, and spleen but not lungs. Prolonged inhibition (14 days, n = 1) caused myocardial ischemic damage and asplenia but not death. Control animals (n = 5) receiving equal doses of a noninhibitory anti-ADAMTS13 mAb remained unaffected. Our results provide evidence for a direct link between TTP and ADAMTS13 inhibition and for a mild disease onset. Furthermore, we present a reliable animal model of this disease as an opportunity for the development and validation of novel treatment strategies.

The humanized anti-glycoprotein Ib monoclonal antibody h6B4-Fab is a potent and safe antithrombotic in a high shear arterial thrombosis model in baboons.

The Fab-fragment of 6B4, a murine monoclonal antibody targeting the human platelet glycoprotein (GP) Ibalpha and blocking the binding of von Willebrand factor (VWF), is a powerful antithrombotic. In baboons, this was without side effects such as bleeding or thrombocytopenia. Recently, we developed a fully recombinant and humanized version of 6B4-Fab-fragment, h6B4-Fab, which maintains its inhibitory capacities in vitro and ex vivo after injection in baboons. We here investigated the antithrombotic properties, the effect on bleeding time and blood loss and initial pharmacokinetics of h6B4-Fab in baboons. The antithrombotic effect of h6B4-Fab on acute platelet-mediated thrombosis was studied in baboons where thrombus formation is induced at an injured and stenosed site of the femoral artery, allowing for cyclic flow reductions (CFRs) which are measured on an extracorporeal femoral arteriovenous shunt. Injection of 0.5 mg/kg h6B4-Fab significantly reduced the CFRs by 80%, whereas two extra injections, resulting in cumulative doses of 1.5 and 2.5 mg/kg, completely inhibited the CFRs. Platelet receptor occupancy, plasma concentrations and effects ex vivo were consistent with what was previously observed. Finally, minimal effects on bleeding time and blood loss, no spontaneous bleeding and no thrombocytopenia were observed. We therefore conclude that h6B4-Fab maintains the antithrombotic capacities of the murine 6B4-Fab, without causing side effects and therefore can be used for further development.

Coronary artery in-stent stenosis persists despite inhibition of the von Willebrand factor--collagen interaction in baboons.

Revascularization techniques, such as angioplasty and stent implantation, frequently lead to restenosis due to the formation of neointima after platelet activation and the concomitant release of various smooth muscle cell mitogenic and attractant factors. We here investigate whether inhibition of initial platelet adhesion after stent implantation can decrease neointima formation in a clinically relevant baboon model of in-stent stenosis using standard treatment with aspirin, clopidogrel and heparin. Inhibition of platelet adhesion was established by administration of the anti-von Willebrand factor (VWF) monoclonal antibody 82D6A3, which inhibits VWF binding to collagen. Administration of 82D6A3 resulted in a complete inhibition of VWF binding to collagen during the first three days after stent implantation. No thrombocytopenia or prolongation of the bleeding time was observed. Our results show that the formation of neointima was not affected in the group of baboons where primary platelet adhesion was abolished with 82D6A3 when compared to the control group. Vascular injury scores were the same in both groups. Inhibition of platelet adhesion during the first three days after stenting, on top of standard treatment with aspirin, clopidogrel and heparin, had no effect on neo-intima formation in a baboon model of in-stent stenosis. During the last decade, attempts to translate seemingly effective therapies based on smaller animal experimentation to the clinic have consistently failed. This study, using a non-human primate model that more closely resembles the clinical situation, presents a model that may be of further clinical interest for studying the prevention of restenosis.

Rational humanization of the powerful antithrombotic anti-GPIbalpha antibody: 6B4.

Fab-fragments of the monoclonal antibody 6B4, raised against human glycoprotein Ibalpha (GPIbalpha), have a powerful antithrombotic effect in baboons by blocking the GPIbalpha binding site for von Willebrand factor (VWF), without significant prolongation of the skin bleeding time. In order to bring this antibody to the clinic,we here humanized for the first time an anti-human GPIbalpha by variable-domain resurfacing guided by computer modeling. First, the genes coding for the variable regions of the heavy and light chains of 6B4 were cloned and sequenced. Based on this, a three-dimensional structure of the Fv-fragment was constructed by using homology-based modeling, and with this and comparison with antibodies with known structure,"murine" putative immunogenic residues which are exposed, were changed for "human-like" residues. The humanized Fab-fragment, h6B4-Fab, was constructed in the pKaneo vector system, expressed and purified and showed in vitro an unaltered, even slightly higher binding affinity for its antigen than the murine form as determined by different ELISA set-ups and surface plasmon resonance. Finally, injection of doses of 0.1 to 1.5 mg/kg of h6B4-Fab in baboons showed that both pharmacokinetics and ex-vivo bio-activity of the molecule were to a large extent preserved. In conclusion,the method used here to humanize 6B4 by resurfacing resulted in a fully active derivative, which is now ready for further development.

Tirofiban versus abciximab: tirofiban is administered at suboptimal dosages when evaluated in an arterial thrombosis model in non-human primates.

To prevent thrombosis in high-risk acute coronary syndrome patients undergoing percutaneous coronary intervention for re-vascularisation, concomitant administration of a glycoprotein IIb/IIIa inhibitor, such as abciximab, tirofiban or eptifibatide, is recommended. Abciximab and eptifibatide are mostly preferred over tirofiban, which is less effective in preventing ischaemic events. We compared the efficacy and bleeding potential of escalating doses of tirofiban and abciximab in non-human primates. The efficacy of tirofiban and abciximab in inhibiting cyclic flow reductions (CFRs) was tested in a high shear arterial thrombosis model. Bleeding was evaluated with the template bleeding time and an incision bleeding model. Abciximab completely inhibited arterial thrombosis after injection of its therapeutic bolus dose. With tirofiban, a dose three times higher than the recommended therapeutic dose caused weak inhibition characterised by a return of CFRs after re-injury. At nine times the recommended therapeutic dose, complete inhibition was observed, and the efficacy of tirofiban was comparable to abciximab at its therapeutic bolus dose. Blood loss was less than with abciximab at its effective dose. In this model, tirofiban compared favourably with abciximab, although only at a dose of 3-9 times the therapeutic dose, and caused less bleeding than abciximab.

Correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects.

BACKGROUND AND OBJECTIVES: Changes in in vitro platelet quality parameters during platelet storage are associated with a decrease of in vivo platelet viability after platelet transfusion. Many attempts have been made to identify the most predictable in vitro parameters for in vivo performance. We used a riboflavin-based ultraviolet (UV) light treatment process designed to inactivate pathogens and white blood cell (WBC) contaminants in blood products as a model system in which to study the correlation of in vitro cell quality with in vivo viability. MATERIALS AND METHODS: Platelet products (n = 18) were collected by a standard Trima apheresis procedure and treated with one of three dose levels of UV light (0, 7.2 or 12.4 J/ml) in the presence of 50 microm riboflavin. Lactate production, glucose consumption and P-selectin expression, pH, pCO(2), pO(2), hypotonic shock response and swirl were measured during 5 days of platelet storage post-UV/RB treatment. Aliquots of these products were radiolabelled on day 5 of storage and were subsequently used to determine platelet recovery and survival time in autologous subjects. RESULTS: The responses of in vitro cell quality were observed to occur in a UV dose-dependent manner. Lactate production and pH were identified as the parameters most strongly correlated with platelet in vivo recovery, which ranged from 5 to 82%. The correlation coefficients (r) for lactate production and pH with in vivo recovery in human subjects were 0.9090 and 0.8831 with P-values of 0.007 and 0.031, respectively. Lactate production and pH were also found to be correlated with platelet survival time, with correlation coefficients of 0.8063 and 0.8384 (the P values were 0.01 and 0.001, respectively). CONCLUSIONS: Using conditions of riboflavin-based UV light treatment, lactate production and pH were identified as having the highest correlations with recovery and survival of radiolabelled platelets in healthy subjects.