Nonclinical Safety Assessment of Anti-Factor D: Key Strategies and Challenges for the Nonclinical Development of Intravitreal Biologics.

PURPOSE: The nonclinical toxicology program described here was designed to characterize the safety profile of anti-factor D (AFD; FCFD4514S, lampalizumab) to support intravitreal (ITV) administration in patients with geographic atrophy (GA). METHODS: The toxicity of AFD was assessed in a single-dose and 6-month repeat-dose study in monkeys at doses up to 10 mg/eye. Toxicity was assessed by clinical ophthalmic examinations, intraocular pressure measurements, ocular photography, electroretinography, fluorescein angiography, optical coherence tomography, and anatomic pathology. RESULTS: Systemic exposure to AFD generally increased with the increase in dose level. The increases in mean maximal concentration and area under the curve values were roughly dose proportional. No accumulation of AFD was observed following 10 doses, and drug exposures were not affected by anti-drug antibodies. AFD was locally and systemically well tolerated in monkeys following ITV doses of up to 10 mg/eye. Ocular effects associated with AFD were limited to transient, reversible, dose-related, aqueous cell responses and injection-related, mild, vitreal cell responses. In the 6-month repeat-dose study, 2 monkeys had a nonspecific immune response to AFD that resulted in severe ocular inflammation, attributed to administration of a heterologous (humanized) protein. CONCLUSIONS: The comprehensive toxicology program in monkeys described here was designed to evaluate the safety profile of AFD and to support multiple ITV injections in the clinic. Administration of a heterologous (humanized) protein presents a challenge, and immunogenicity in nonclinical species is not predictive of immunogenicity in humans. Taken together, the results of the nonclinical program described here support the use of AFD in patients with GA.

Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level.

The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action.

A microchip flow-chamber system for quantitative assessment of the platelet thrombus formation process.

As the pathogenesis of arterial thrombosis often includes platelet thrombus formation (PTF), antiplatelet agents are commonly used for the prevention of thromboembolic events. Here, using a novel microchip flow-chamber system we developed to quantitatively analyze the PTF process, we evaluated the pharmacological efficacies of antiplatelet agents under different arterial shear rates. Hirudin-anticoagulated whole blood was perfused over a collagen-coated microchip at shear rates of 1000, 1500, and 2000s(-1), and PTF in the absence and presence of various antiplatelet agents was observed microscopically and quantified by measuring flow-pressure changes. The onset of PTF was measured as T(10) (time to reach 10 kPa), and AUC(10) (area under the flow pressure curve for the first 10 min) was calculated to quantify the overall stability of the formed thrombus. Aspirin and AR-C66096 (P2Y(12)-antagonist) at high concentrations (50 µM and 1000 nM, respectively) prolonged T(10) only modestly (AR-C66096>aspirin), but effectively decreased AUC(10), resulting in unstable PTF at all examined shear rates. With dual inhibition using both aspirin (25 µM) and ARC-66096 (250 nM), AUC(10) was drastically reduced. Nearly complete suppression of AUC(10) was also observed with abciximab (2 µg ml(-1)) and beraprost (PGI(2)-analog; 4 nM). Although OS-1 (GPIbα-antagonist; 100 nM) prevented complete capillary occlusion, significant amounts of microscopic thrombi were observed on the collagen surface. In contrast to abciximab and beraprost, OS-1 differentially affected PTF under higher shear conditions. Our novel analytical system is capable of distinguishing the pharmacological effects of various antiplatelet agents under physiological shear rates, suggesting that this system may aid in the determination of the appropriate type and dose of antiplatelet agent in the clinical setting.

Comparación entre dos métodos de producción para la elaboración de antivenenos ofídicos / Snake antivenin: Comparison between two production methods

Las mordeduras producidas por serpientes venenosas son un serio problema médico en varias regiones del mundo y sobre las cuales los sistemas de salud actúan en diferentes grados en lo referente a tratamiento y prevención. Sin embargo, el tratamiento de las mordeduras de serpientes venenosas en animales domésticos puede resultar difícil por diversos motivos, siendo uno de estos la baja oferta o ausencia de antivenenos para uso veterinario. Las presiones comerciales en la industria farmacéutica han llevado a una reducción en la producción de antivenenos en varias partes del mundo, su disponibilidad es, a veces, bastante limitada y en algunos casos, son imposibles de conseguir. En este trabajo, inmunizamos caballos con veneno de serpientes Sudamericanas para obtener el plasma hiperinmune que fue procesado para obtener IgG entera o fragmentos F(ab´)2 usando dos métodos convencionales (fraccionamiento por ácido caprílico o doble precipitación salina y digestión con pepsina). Los antivenenos así obtenidos fueron probados en sus características bioquímicas e inmunoquímicas, así como en su potencia neutralizante. El SDS-PAGE de los antivenenos mostró bandas en el orden de los 150 y 100 kDa en los antivenenos conteniendo IgG entera o fragmentos F(ab´)2, respectivamente. La presencia de albúmina o contaminantes de alto o bajo peso molecular no fue detectada en ninguna de las preparaciones. No se observaron diferencias importantes en la potencia neutralizante de los antivenenos, aunque el costo de producción fue mucho más bajo en la obtención de IgG completa. A partir de esto, se sugiere que los bajos costos de producción en la obtención de antivenenos de IgG entera para uso veterinario, hacen a esta tecnología adecuada y rentable cuando la producción de F(ab´)2 no es posible.

Bites by venomous snakes are a serious medical problem in several regions of the world, on which the different health systems act with different modalities. Nevertheless, the treatment of venomous snakebites in domestic animals can turn difficult due several problems among which, the conspicuous, is the low availability or lack of antivenoms for veterinary use. As commercial pressures on the pharmaceutical industry have led to a reduction in the production of antivenins in several parts of the world, their availability is sometimes rather limited and sometimes these products are impossible to obtain. In this work, we immunized horses with venom of South American vipers to obtain hyperimmune plasma. The plasma was processed to separate whole IgG of F(ab´)2 fragments using two conventional methods (caprylic acid fractionation or double saline precipitation and pepsin digestion). The obtained antivenins were tested for their biochemical and immunochemical characteristics and neutralizing potency. The SDS-PAGE of the antivenins showed, in the processed antivenin, bands in the order of 150 and 100 kDa in the whole IgG or F(ab´)2 fragments, respectively. The presence of albumin or contaminants of high or low molecular weight was not detected in any of the preparations. No important differences were observed in the neutralizing potency of the antivenins, although production cost was very low with the method used to obtain pure IgG. The low production cost makes the production of antivenins for veterinary use profitable when the production of F(ab´)2 fragments is not possible.

Certolizumab pegol.

Certolizumab pegol (Cimzia(®)) is currently the only PEGylated anti-TNFα biologic approved for the treatment of rheumatoid arthritis and Crohn disease. The product, developed by UCB, is a humanized antigen-binding fragment (Fab') of a monoclonal antibody that has been conjugated to polyethylene glycol. Certolizumab pegol was approved as a treatment for rheumatoid arthritis in the EU, US and Canada in 2009, and as a treatment for Crohn disease in Switzerland in 2007 and the US in 2008. Certolizumab pegol is entering into an increasingly competitive marketplace, especially in rheumatoid arthritis, but clinical data demonstrate benefits across a range of clinical, radiographic and patient reported outcomes.

Application of ex vivo flow chamber system for assessment of stent thrombosis.

OBJECTIVE: Factors influencing platelet accumulation around stents were to be investigated by an ex vivo flow chamber system. METHODS AND RESULTS: Platelet accumulations on collagen surfaces under flow conditions were augmented in the presence of stents, especially at sites downstream from coil stents. Densitometric analysis revealed that 4.9+/-0.8 times more platelets accumulated downstream from coil stents than were formed downstream from tube stents (P<0.01), suggesting that stent morphology is an important determinant factor of its thrombogenicity. Platelet accumulations around stents were significantly inhibited by a combination of ticlopidine and aspirin, whereas aspirin alone produced only modest inhibition. Anti-glycoprotein IIb/IIIa (abciximab) inhibited platelet accumulation around stents in a dose-dependent manner, whereas the antibody blocking von Willebrand factor binding to glycoprotein Ib(alpha), which had been shown to inhibit platelet thrombus formation under high shear rates, did not inhibit the accumulation downstream from the coil stents. Our results suggest that the important characteristics of in vivo stent thrombosis, ie, augmented platelet accumulation with coil stents and the strong antithrombotic effect of the combination antiplatelet agents and an anti-glycoprotein IIb/IIIa, can be reproduced in ex vivo perfusion model. CONCLUSIONS: We conclude that an ex vivo perfusion system is useful in the assessment of the thrombogenicity of various stents and in the screening of effective antiplatelet agents.

Use of abciximab (c7E3 Fab, ReoPro) as an adjunct to balloon angioplasty.

OBJECTIVE: To estimate the magnitude of the clinical benefits that may result from use of abciximab at the time of angioplasty and the cost of achieving them. DATA SOURCES: Four published randomized control trials. DATA SYNTHESIS: Meta-analysis of outcomes at six months. RESULTS: Use of abciximab in comparable high risk populations, in the manner described in these trials, is estimated to have the following effects: It does nto influence mortality within the first six months. It reduces the rate of myocardial infarction (MI) by 3.3/100 treatments with a 95% CI of 1.6 to 5.2. It may reduce the need for revascularization (angioplasty or coronary artery bypass graft) by 2.1/100 treatments (95% CI -1.0 to 5.0). It does not cause any significant increase in major hemorrhagic events. There is no evidence that it influences restenosis rates. The net cost per MI prevented would be approximately $44,000, ranging from approximately $29,000 to $71,000 on sensitivity analysis. The net cost per adverse event prevented (MI plus revascularization procedure) would be approximately $27,000 (sensitivity analysis $16,000 to $57,000). Use of abciximab for all of the approximately 17,487 angioplasties carried out in Canada each year may prevent 395 myocardial infarcts and 186 revascularization procedures, at an overall cost of approximately $29 million and a cost effectiveness of approximately $50,000 per adverse event prevented. (This assumes the same proportional reduction in events as in these four studies, and that 35% of procedures are high risk). SIGNIFICANCE: Possible eventual prolongation of life due to fewer periprocedural MIs with abciximab use cannot be quantified. Thus, these estimates of cost effectiveness cannot be used to compare this intervention directly with others in terms of dollars per life year saved. The field is evolving rapidly and these conclusions may soon have to be modified. Increasing use of stents will probably slightly reduce, but not abolish, the health benefits of abciximab use. These estimates are based on only four trials. However, until more trials are completed they provide the best available evidence on which to base policy decisions.

A risk-benefit assessment of abciximab in angioplasty.

Advances in percutaneous coronary intervention (PCI) have allowed procedures to be performed on a variety of patients with a spectrum of challenging coronary anatomy. Abciximab has permitted further expansion and has made the procedure safer. Abciximab is a chimerised murine monoclonal antibody directed against the platelet glycoprotein (GP) IIb-IIIa receptor. Binding to this receptor inhibits platelet aggregation to a wide variety of biological agonists. It also binds to alphavbeta3 and leucocyte MAC-1 receptors; the biological significance of its affinity to these receptors is unclear. Abciximab has an extremely short plasma half-life. Since abciximab binds to the platelet GP IIb-IIIa receptor with great avidity it has an extremely long biological half-life. The use of abciximab is currently confined primarily to PCIs. The first large trial, EPIC, established that abciximab, given with aspirin (acetylsalicylic acid) and heparin, reduced the frequency of peri-procedural ischaemic events by 35% in high-risk patients. For this reduction a bolus of 0.25 mg/kg was followed by a 12-hour infusion of abciximab. However, the transfusion rate was doubled. A subsequent trial, EPILOG, indicated that reduction of the dose of heparin along with expeditious removal of arterial access sheaths, reduced the rate of haemorrhagic complications to a level comparable with placebo-treated patients. while also amplifying the reduction in ischaemic events. In a third trial, EPISTENT, this benefit was shown to include patients undergoing elective coronary stent implantation. Additional trials have demonstrated that the same effect is present in patients undergoing primary PCI for acute myocardial infarction and in patients undergoing PCI for refractory unstable angina pectoris. In the latter situation, treatment with abciximab for 18 to 24 hours preceding the intervention reduced the rate of myocardial infarction even before the procedure was begun. The rationale for the use abciximab is thus clearly established. Bleeding complications can be reduced by limiting the heparin dose, avoiding unneeded venous access site punctures, and expeditious removal of arterial sheaths. In emergency coronary artery bypass surgery, platelet transfusion reduces the number of receptors occupied per platelet and is likely to reduce the degree of postoperative bleeding. The cost of abciximab remains an issue; however, this is partially offset by the reduction in ischaemic complications and accompanying resource use. In patients undergoing elective coronary stenting, abciximab use reduced the long term rate of target vessel revascularisation. The degree to which this reduction results in further cost savings will require further analysis.

[Medico-economic evaluation of abciximab]. / Evaluation médico-économique d'abciximab.

Abciximab (RéoPro) is a chimeric monoclonal antibody directed against platelet glycoprotein IIb-IIIa. It reduces the frequency of major cardiac ischaemic events after percutaneous transluminal coronary angioplasty (PTCA). This treatment is more expensive than the commonly used treatment (aspirin and heparin). However, a treatment can only be validly assessed in term of its cost-benefit ratio. The authors present the results of a medico-economic evaluation of abciximab in the French institutional context, based on the three-year clinical results of the EPIC study (Evaluation of abciximab for the Prevention of Ischemic Complications). The efficacy of treatment was expressed by means of two quantitative indicators: the number of patients with no major ischaemic event (MIE) and the number of years of life without myocardial infarction or revascularization. According to French good practice recommendations for economic evaluation of therapeutic strategies, major ischaemic events were assessed by using the complete cost of the corresponding homogeneous groups of diseases (GHM). Medico-economic evaluation of abciximab showed that the net excess cost per patient for national health insurance was 395 Francs, while the cost of the product is 5,300 Francs. The incremental cost per additional patient with no major ischaemic event is 6,585 Francs and the incremental cost per additional year of life without myocardial infarction or revascularization is 2,469 F. From a public health point of view, abciximab administered by bolus injection plus a 12-hour infusion, presents a good cost-effectiveness ratio in the prevention of cardiac ischaemic complications in high-risk patients after PTCA.

Preclinical assessment of immunoreactivity of a new purified equine F(ab')2 against European viper venom.

The immunological and pharmacokinetic properties of a new, further purified, pasteurized preparation of equine F(ab')2 (VIPERFAV) against Vipera aspis, Vipera berus, and Vipera ammodytes venom were compared with the current equine F(ab')2 preparation (IPSER Europe). Affinity constants of the V. aspis-specific F(ab')2 were determined using biosensor technology and found to be in the range of 10(8) M-1 for the four antigenic fractions of V. aspis toxins and for both F(ab')2 preparations. The improvement of 51% in the specific activity (LD50 mg-1) of the new F(ab')2 was in close agreement with the 1.8-fold increase in the immunoreactive fraction of the new preparation. In vivo investigations of venom immunocomplexation by F(ab')2 in rabbits confirmed the ability of F(ab')2 to neutralize and redistribute toxin venom. Infusion of a stoichiometric molar ratio (i.e., 1 mg kg-1) of the new antivenom induced a 2.3-fold elevation of the plasma venom concentration with a Tmax observed 8 h after F(ab')2 administration and a decline in the terminal half-life from 31.92 +/- 4.49 h to 16.73 +/- 4.34 h, in contrast, for the venom alone. The area under the curve was 1.4-fold greater in the VIPERFAV group than in the IPSER Europe group during the post-F(ab')2 infusion period. Increasing the F(ab')2 dose to 3 mg kg-1 increased by 27% the percent of venom bound to F(ab')2. Finally, the greater the venom distribution, the smaller and less pronounced the plasma redistribution. These results demonstrate that the purification and pasteurization steps involved in the preparation of the new F(ab')2 have no deleterious influence on F(ab')2 affinity but, on the contrary, improve the protective efficacy. Alteration of viper venom kinetics by specific F(ab')2 antivenom was also shown to be dependent on the interval between of F(ab')2 administration and venom bite and on the specific F(ab')2 dose administered.

Rapid assessment of platelet function with a modified whole-blood aggregometer in percutaneous transluminal coronary angioplasty patients receiving anti-GP IIb/IIIa therapy.

BACKGROUND: The glycoprotein (GP) IIb/IIIa receptor antagonist abciximab (c7E3 Fab, ReoPro) is approved for use in high-risk percutaneous transluminal coronary angioplasty (PTCA). At present, no "point of care" exists for measuring pharmacological GP IIb/IIIa blockade. To address this need, the Chrono-log Whole Blood Aggregometer, which measures platelet aggregation by electrical impedance, was adapted to test platelet function at the bedside. METHODS AND RESULTS: GP IIb/IIIa receptor blockade, impedance (5 microg/mL collagen), and turbidimetric aggregation (5 and 20 micromol/L ADP) measurements were obtained on 14 PTCA patients who received the standard bolus plus a 12-hour infusion of abciximab. During abciximab administration, mean GP IIb/IIIa receptor blockade was > 91%, and both impedance and turbidimetric aggregation were inhibited by > or = 90%. At 12 hours after abciximab treatment, the mean inhibition of turbidimetric platelet aggregation to 5 and 20 micromol/L ADP was 65+/-20% and 49+/-14%, respectively, and inhibition of impedance aggregation was 69+/-12%. GP IIb/IIIa receptor blockade was 67+/-8%. At 36 hours after abciximab treatment (n=8), the mean inhibition of turbidimetric platelet aggregation to 5 and 20 micromol/L ADP was 44+/-21% and 30+/-14%, respectively, whereas impedance aggregation was inhibited by 60+/-14%. GP IIb/IIIa receptor blockade was 57+/-7%. CONCLUSIONS: During and at 12 hours after abciximab therapy, impedance and turbidimetric platelet aggregation to 5 micromol/L ADP were comparable and closely correlated with GP IIb/IIIa receptor blockade. However, at 36 hours after abciximab treatment, impedance platelet aggregation more closely paralleled GP IIb/IIIa receptor blockade and indicated a slower recovery of platelet function than turbidimetric aggregometry.

Assessment of an ovine antivenom raised against venom from the desert black cobra (Walterinnesia aegyptia).

The desert black cobra (Walterinnesia aegyptia) is an elapid widely distributed throughout the deserts of Saudi Arabia and currently available antivenoms are ineffective in the treatment of its envenoming. Walterinnesia aegyptia venom was assessed for several of its physicochemical, enzymatic and biological characteristics. An antivenom was raised in sheep using a low-dose immunization schedule and digested with papain to provide Fab fragments. The antivenom neutralized all of the above enzymatic and biological activities and provided good protection in mice (ED50 0.25 g/kg), whereas the commercial polyspecific products showed only partial neutralization and did not protect mice.